New peptide, process for preparation thereof and use thereof

ABSTRACT

The invention relates to compounds and salts of compounds which are diaminopimelyl peptides, N-acetoxypropionyl or N-t-butoxycarbonyl-amino-acid - (α-OBzl) glutamic acid peptides or N-acylated derivatives of (α-OBzl) glytamic acid or of (α-OBzl) aminoadipic acid. 
     The compounds and their salts are starting materials for the preparation of pharmacologically active peptides.

This application is a division of Ser. No. 377,841, filed May 13, 1982,which is a division of application Ser. No. 229,072, filed Jan. 28,1981, now U.S. Pat. No. 4,349,466, which is in turn acontinuation-in-part of application Ser. No. 201,241 filed Oct. 27,1980, now U.S. Pat. No. 4,322,341, which is in turn acontinuation-in-part of application Ser. No. 171,024 filed July 22,1980, now abandoned, which is a continuation-in-part of application Ser.No. 149,441, filed on May 13, 1980, now abandoned, which is acontinuation-in-part of application Ser. No. 147,710, filed on May 8,1980, now abandoned, which is a continuation-in-part of application Ser.No. 110,020, filed on Jan. 7, 1980, now abandoned, which is acontinuation-in-part of application Ser. No. 093,523 filed on Nov. 13,1979, now U.S. Pat. No. 4,311,640, Jan. 19, 1982.

This invention relates to a new peptide. More particularly, thisinvention relates to a new peptide and the pharmaceutically acceptablesalt thereof, which have pharmacological activities, to processes forthe preparation thereof and to a new intermediate for preparing theactive peptide, and to the pharmaceutical composition comprising thesame and a method of use thereof.

Firstly, it is to be noted that this invention is originated from andbased on the first and new discovery of the new active peptide, i.e.FR-900156 substance. That is, the FR-900156 substance was firstly andnewly isolated in pure form from a culture broth obtained byfermentation of a new strain belonging to the genus Streptomyces andcharacterized by the physico-chemical properties.

Thereafter, as a result of extensive study, the inventors of thisinvention succeeded in determining the chemical structure thereof bycommanding physical and chemical techniques so that they could give thesequential structure of the formula (If) indicated below to FR-900156substance and proposed with a firm belief the possible stereoisomericstructure of the formula (Ie) as indicated below for the same. ##STR1##

Further, after the structural elucidation of FR-900156 as explainedabove, the inventors of this invention have continued extensive studieson total synthesis of the compound of the formula (If) including thecompound of the formula (Ie) so that they have succeeded in completingthe industrially advantageous and applicable synthetic processes forpreparation of the same, and further have syntherized a lot of relatedcompounds.

A new peptide of this invention is represented by the following formula(I): ##STR2## wherein

R¹ is hydrogen or acyl;

R_(b) ¹ is hydrogen, methyl, isopropyl, hydroxymethyl, protectedhydroxymethyl or benzyl;

R² and R^(q) are each hydrogen, carboxy, protected carboxy, or a groupof the formula: ##STR3## wherein

R_(a) ² is mono- or di-carboxy (or protected carboxy) lower alkyl orar(carboxy or protected carboxy) lower alkyl whose aryl moiety may besubstituted by hydroxy,

R_(b) ² is hydrogen or lower alkyl;

R^(p) is hydrogen, carboxy, protected carboxy with proviso that when oneof R² and R^(q) is hydrogen, then the other is carboxy or protectedcarboxy or a group of the formula: ##STR4## wherein R_(a) ² and R_(b) ²are each as defined above;

R^(r) is hydrogen or amino protective group,

m is an integer 1 to 3; and

n is an integer 0 to 2, provided that when R¹ is hydrogen,t-butoxycarbonyl or N-acetylmuramyl,

R_(b) ¹ is methyl, m is an integer 2 and n is an integer 1, then

R² is hydrogen, protected carboxy or a group of the formula: ##STR5##wherein R_(a) ² is mono- or di-carboxy (or protected carboxy) loweralkyl having 1 and 3 to 6 carbon atoms, α-carboxyethyl, α-protectedcarboxyethyl, ar-(carboxy or protected carboxy) lower alkyl whose arylmoiety may be substituted by hydroxy and R_(b) ² is as defined above.

Particulars of the various definitions, which are mentioned hereinabove,and hereinafter and preferred examples thereof are explained in thefollowing.

The term "lower" is intended to mean a group having 1 to 6 carbonatom(s), unless otherwise provided.

(1) Re. Acyl for R¹ and R_(c) ¹ :

Generally, "acyl" may be an acyl group derived from an acid such asorganic carboxylic acid, carbonic acid, or carbamic acid, or the thioacid or imidic acid corresponding to each of the preceeding acids, or anorganic sulfonic acid, each of which includes an aliphatic, an aromaticand/or a heterocyclic groups in its molecule; carbamoyl; orcarbamimidoyl.

Suitable examples of said acyl are illustrated below.

Aliphatic acyl means an acyl group derived from an aliphatic acidincludes:

alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl,isovaleryl, pivaloyl, hexanoyl, α-ethylhexanoyl, heptanoyl, lauroyl,stearoyl, docosanoyl, 2-heneicosylpentacosanoyl, hexacosanoyl[CH₃(CH₂)₂₄ C0], tetrapentacontanoyl[CH₃ (CH₂)₅₂ CO],2-hexadecyloctadecanoyl[[CH₃ (CH₂)₁₅ ]₂ CHCO], n-tetracosanoyl[CH₃(CH₂)₂₂ CO], 2-docosyltetracosanoyl[[CH₃ (CH₂)₂₁ ]₂ CHCO], etc.);

cycloalkanecarbonyl (e.g. cyclopentanecarbonyl, cyclopropanecarbonyl,cyclohexanecarbonyl, cycloheptanecarbonyl, etc.);

bridgedalicycliccarbonyl such as bicyclic or tricyclic alkane oralkenecarbonyl (e.g. norbornane carbonyl, adamantanecarbonyl, etc.);

alkenoyl (e.g. acryloyl, methacryloyl, crotonoyl, oleoyl, arachidonyl,etc.);

lower alkylthio(lower)alkanoyl (e.g. methylthioacetyl, ethylthioacetyl,etc.);

lower alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, t-pentoxycarbonyl,etc.);

lower alkanesulfonyl(lower)alkoxycarbonyl (e.g. 2-(mesyl)ethoxycarbonyl,etc.);

lower alkylthio(lower)alkoxycarbonyl (e.g. 2-(ethylthio)ethoxycarbonyl,etc.);

lower alkenyloxycarbonyl (e.g. allyloxycarbonyl, etc.);

alkatetraenoyl (e.g. 3,7-dimethylnone-2,4,6,8-tetraenoyl, etc);

lower alkynyloxycarbonyl (e.g. 1,1-dimethylpropargyloxycarbonyl, etc.);

lower alkylcarbamoyl (e.g. methylcarbamoyl, etc.);

(N-lower alkyl)thiocarbamoyl [e.g. (N-methyl)thiocarbamoyl, etc.];

lower alkylcarbamimidoyl (e.g. methylcarbamimidoyl, etc.);

oxalo;

alkoxalyl (e.g. methoxalyl, ethoxalyl, propoxalyl, etc.);

In the above exemplified aliphatic acyl, the aliphatic hydrocarbonmoiety, particularly the alkyl group and alkane moiety and the alkenylgroup and alkene moiety may have optionally one or more suitablesubstituent(s) such as amino, halogen (e.g. fluorine, chlorine, bromine,etc.), hydroxy, hydroxyimino, carboxy, lower alkoxy (e.g. methoxy,ethoxy, propoxy, etc.), lower alkoxycarbonyl, acylamino such as loweralkanoylamino (e.g. acetylamino, propionylamino, etc.),ar(lower)alkoxycarbonylamino (e.g. benzyloxycarbonylamino, etc.), etc.or acyloxy such as lower alkanoyloxy (e.g. acetoxy, propoxy, etc.),ar(lower)alkanoyloxy (e.g. benzylcarbonyloxy, etc.), aroyloxy (e.g.benzoyloxy, etc.), or 5- or 6-membered cycloalkenyl (e.g.1,3,3-trimethyl-1-cyclohexen-2-yl, etc.).

Preferred examples of aliphatic acyl having such substituents may beexemplified by

hydroxyalkanoyl (e.g. glycoloyl, lactoyl, 2-hydroxybutyl, mycoloyl,##STR6## etc.,

lower alkoxy(lower)alkanoyl (e.g. methoxyacetyl, 2-methoxypropionyl,etc.),

carboxy(lower)alkanoyl (e.g. carboxyacetyl, carboxypropionyl, etc.),

(lower)alkoxycarbonyl(lower)alkanoyl (e.g. methoxalyl, ethoxalyl,methoxycarbonylacetyl, propoxycarbonylpropionyl, ethoxycarbonylbutyryl,etc.),

halo(lower)alkoxycarbonyl (e.g. chloromethoxycarbonyl,tribromoethoxycarbonyl, trichloroethoxycarbonyl, etc.),

6-membered cycloalkenyl-alkatetraenoyl (e.g. retinoyl, etc.),

and the like.

Aromatic acyl means an acyl group derived from an acid havingsubstituted or unsubstituted aryl group, in which the aryl group mayinclude phenyl, tolyl, xylyl, naphthyl and the like, and suitableexamples thereof are illustrated as follows:

aroyl (e.g. benzoyl, toluoyl, xyloyl, naphthoyl, phthaloyl, etc.);

ar(lower)alkanoyl (e.g. phenylacetyl, diphenylacetyl, etc.);

ar(lower)alkenoyl (e.g. cinnamoyl, etc.);

aryloxy(lower)alkanoyl (e.g. phenoxyacetyl, etc.);

arylthio(lower)alkanoyl (e.g. phenylthioacetyl, etc.);

arylamino(lower)alkanoyl (e.g. N-phenylglycyl, etc.);

arenesulfonyl (e.g. benzenesulfonyl, tosyl, naphthalenesulfonyl, etc.);

aryloxycarbonyl (e.g. phenoxycarbonyl, tolyloxycarbonyl, etc.);

aralkoxycarbonyl (e.g. benzyloxycarbonyl, benzhydryloxycarbonyl,trityloxycarbonyl, α-naphthylmethoxycarbonyl, etc.);

arylcarbamoyl (e.g. phenylcarbamoyl, tolylcarbamoyl, naphthylcarbamoyl,etc.);

arylglyoxyloyl (e.g. phenylglyoxyloyl, etc.);

arylthiocarbamoyl (e.g. phenylthiocarbamoyl, etc.);

arylcarbamimidoyl (e.g. phenylcarbamimidoyl, etc.); and the like.

In the above exemplified aromatic acyl, the aromatic hydrocarbon moiety(particularly, aryl moiety) and/or aliphatic hydrocarbon moiety(particularly, alkane moiety) may have optionally one or more suitablesubstituent(s), such as the same as those exemplified as the suitablesubstituent for alkyl group and alkane moiety as mentioned above.

Preferred example of aromatic acyl having such substituents may beexemplified by

hydroxyaroyl (e.g. salicyloyl, etc.)

haloaroyl (e.g. chlorobenzoyl, etc.),

haloar(lower)alkanoyl (e.g. chlorophenylacetyl, etc.),

hydroxyar(lower)alkanoyl (e.g. mandelyl, etc.), methoxyaralkanoyl [e.g.10-(2,3,4-trimethoxy-6-methylphenyl)-decanoyl, etc].

Heterocyclic acyl means an acyl group derived from an acid havingheterocyclic group and includes:

heterocyclic carbonyl, in which the heterocycle moiety is 5 to 6membered heterocycle containing at least one hetero atom selected fromnitrogen, oxygen and sulfur (e.g. thenoyl, furoyl, pyrrolecarbonyl,5-oxo-2-pyrrolidinecarbonyl, nicotinoyl, etc.);

heterocyclo(lower)alkanoyl, in which the heterocycle moiety is 5 to 6membered heterocycle containing at least one hetero atom selected fromnitrogen, oxygen and sulfur (e.g. thienylacetyl, furylacetyl,imidazolylpropionyl, tetrazolylacetyl,2-(2-amino-4-thiazolyl)-2-methoxyiminoacetyl, N-acetylmuramyl, etc.);and the like.

In the above exemplified heterocyclic acyl, heterocycle moiety and/orthe aliphatic hydrocarbon moiety may have optionally one or moresuitable substituent(s) such as the same as those exemplified as thesuitable substituent for aliphatic acyl as mentioned above.

Further, in the above exemplified acyl, in case that these acyls haveone or more functional group such as hydroxy, amino, carboxy, and thelike, such groups may be protected by conventional protective groups(s).

(2) Re. Protected carboxy for R², R^(p), R₁ ^(p) and R^(q) :

A protective group of the protected carboxy includes a conventionalprotective group for tentatively protecting a carboxy group which isconventionally used in the field of amino acid and peptide chemistry.

As preferred examples of protected carboxy, there may be exemplified anester such as an ester with silyl compound (hereinafter referred to assilyl ester), an ester with an aliphatic hydroxy compound (hereinafterreferred to as aliphatic ester) and an ester with a hydroxy compoundcontaining an aromatic group (hereinafter referred to as aromaticester), and a protected carbazoyl of the formula: --COHNNHY (wherein Yis hydrogen or an amino protective group).

Concrete examples of such a protected carboxy are exemplified asfollows:

suitable silyl ester such as trialkylsilyl (e.g. trimethylsilyl,triethylsilyl, etc.) ester,

halo-alkylsilyl (e.g. chloro-dimethylsilyl, dichloro-methylsilyl, etc.)ester,

trihalosilyl (e.g. trichlorosilyl, etc.) ester,

alkylalkoxysilyl (e.g. methyl-diethoxysilyl, etc.) ester,

trialkoxysilyl (e.g. tris(2-chloroethoxy)silyl, etc.) ester, and thelike;

suitable aliphatic hydrocarbon ester such as alkyl (e.g. methyl, ethyl,propyl, isopropyl, butyl, tertbutyl, etc.) ester,

cycloalkyl (e.g. cyclopentyl, cyclohexyl, etc.) ester and the like; and

suitable ester containing an aromatic group such as aryl (e.g. phenyl,tolyl, xylyl, etc.) ester,

aralkyl (e.g. benzyl, diphenylmethyl, phenethyl, etc.) ester,

aryloxyalkyl (e.g. phenoxymethyl, phenoxyethyl, etc.) ester,

aroylaklyl (e.g. phenacyl, toluoylethyl, etc.) ester, and the like.

The ester forming group (e.g. substituted silyl, aliphatic hydrocarbonresidue, aryl, aralkyl, aryloxyalkyl, aroylalkyl and the like, asexemplified above) may optionally have one or more appropriatesubstituent(s) such as alkyl (e.g. methyl, ethyl, etc.), cycloalkyl(e.g. cyclopropyl, cyclohexyl, etc.), alkoxy (e.g. methoxy, ethoxy,etc.), alkanoyloxy (e.g. acetoxy, etc.), alkylthio (e.g. methylthio,etc.), halogen (e.g. chlorine, etc.), cyano, nitro, etc.

Examples of such substituted esters may be mono(di or tri)haloalkyl(e.g. chloromethyl, bromoethyl, dichloromethyl, 2,2,2-trichloroethyl,2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, etc.) ester, cyanoalkyl (e.g.cyanomethyl, cyanoethyl, etc.) ester, cycloalkyl-substituted-alkyl (e.g.1-cyclopropylethyl, etc.) ester, mono(di, tri, tetra or penta)halophenyl(e.g. 4-chlorophenyl, 3,5-dibromophenyl, 2,4,5-trichlorophenyl,2,4,6-trichlorophenyl, pentachlorophenyl, etc.) ester, and the like.

With regard to the term "protected carboxy" for R², R^(p), R₁ ^(p) andR^(q), it is to be understood that this group bear the meaning not onlyin synthetic manufacture of the object compound by chemical process(es),but also in physiological and pharmaceutical properties of the objectcompound per se.

That is, in the meaning of the synthetic manufacture, free carboxy groupfor R², R^(p), R₁ ^(p) and R^(q) may be transformed into the "protectedcarboxy" group as mentioned above before conducting the process(es) forpreventing any possible undesired side reaction(s), and the "protectedcarboxy" group in the resultant compound may be transformed into freecarboxy group after the reaction is conducted. This will be apparentfrom the explanation of the processes in the following.

On the other hand, in the meaning of the physiological andpharmaceutical properties of the object compound, the compound bearingthe "protected carboxy" group for R², R^(p), R₁ ^(p) and/or R^(q) isoptionally used for the physiologically and pharmaceutically activecompound per se. More particularly, as such a protected carboxy group,"esterified carboxy" group is optionally given as a preferable example,including a conventional esterified carboxy. As suitable examples of the"esterified carboxy", there may be exemplified the same esterifiedcarboxy as illustrated above for explanation of the protected carboxy,among which, as preferred examples, there may be given an aliphatichydrocarbon ester such as alkyl ester, i.e. alkoxycarbonyl (e.g.methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, t-butoxycarbonyl, etc.) and the like.

(3) Re. A group of the formula: ##STR7##

Suitable example of lower alkyl for R_(b) ² and lower alkyl moiety ofmono- or di-carboxy(lower)alkyl and of ar(carboxy)lower alkyl for R_(a)² is one having 1-6 carbon atoms such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, isopentyl and the like.

Suitable examples of aryl moiety of ar(carboxy)lower alkyl for R_(a) ²is phenyl, tolyl, xylyl, naphthyl and the like. In this respect, it isto be noted that "ar(carboxy)lower alkyl" can alternatively expressed bythe wording "lower alkyl" which is substituted by carboxy and arylgroup.

Carboxy in "mono- or di-carboxy lower alkyl" for R_(a) ² and"ar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy"for R_(a) ² may be protected by a conventional protective group; namely,R_(a) ² includes within its definition also protected carboxy-loweralkyl and ar(protected carboxy)lower alkyl which may be substituted byhydroxy. Suitable example of such a protected carboxy is the same asthat exemplified for R^(p) and R^(q).

Most preferred examples of a group of the formula: ##STR8## areillustrated as follows: ##STR9## the corresponding group, in which thecarboxy group is protected by a conventional carboxy protective group.

(4) Re. Protected hydroxymethyl for R_(b) ¹ :

Hydroxy group of hydroxymethyl for R_(b) ¹ may be protected by aconventional hydroxy protective group; namely, R_(b) ¹ includesprotected hydroxymethyl.

Some preferred examples of the hydroxy protective group may beexemplified by an acyl such as substituted or unsubstituted alkanoyl(e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl,bromoacetyl, dichloroacetyl, trifluoroacetyl, etc.), substituted orunsubstituted aroyl (e.g. benzoyl, toluoyl, xyloyl, nitrobenzoyl,bromobenzoyl, salicyloyl, etc.), arylalkyl (e.g. benzyl) or the like.

(5) Re. Amino protective group for R^(r), R₁ ^(r) and Y:

The amino protective group includes a conventional protective group fortentatively protecting an amino group, which is used in the field ofamino acid and peptide chemistry. That is, in the peptide synthesis, itis understood that, for bonding a desired "reactive" amino group (--NH₂)with a desired "reactive" carboxy group (--COOH) to form a desiredpeptide bond (--CONH--) between them, it is preferable to tentativelyprotect the other undesired "reactive" amino group to convert it into anunreactive or less reactive protected amino group in the reaction inorder to avoid the side reaction between the undesired "reactive" aminogroup and desired "reactive" carboxy groups. Further, it is understoodthat it is preferable that a protective group in such protected aminogroup is easily eliminable according to the necessity in the posttreatment of the object peptide. Accordingly, an amino protective groupto meet the above requirements can be used and suitable one should beselected according to the kind and property of the component to be usedin this invention.

As preferred examples of the amino protective group, the followingexamples are illustrated:

Acyl, particularly organic acyl, for example, substituted orunsubstituted aliphatic hydrocarbon-oxycarbonyl such as

alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,butoxycarbonyl, t-butoxycarbonyl, t-pentoxycarbonyl, etc.),

haloalkoxycarbonyl (e.g. chloromethoxycarbonyl, tribromoethoxycarbonyl,trichloroethoxycarbonyl, etc.),

an alkane- or arene-sulfonylalkoxycarbonyl (e.g.2-(mesyl)ethoxycarbonyl, 2-(p-toluenesulonyl)ethoxycarbonyl, etc.),

an alkylthio- or arylthioalkoxycarbonyl (e.g.2-(ethylthio)ethoxycarbonyl, 2-(p-tolylthio)ethoxycarbonyl, etc.),substituted or unsubstituted alkanoyl such as halo(lower)alkanoyl (e.g.formyl, trifluoroacetyl, etc),

a monocyclic or fusedcyclic-alicyclic oxycarbonyl (e.g.cyclohexyloxycarbonyl, adamantyloxycarbonyl, isobornyloxycarbonyl,etc.),

substituted or unsubstituted alkenyloxycarbonyl (e.g. allyoxycarbonyl,etc.),

substituted or unsubstituted alkynyloxycarbonyl (e.g.1,1-dimethylpropargyloxycarbonyl, etc.) or the like,

substituted or unsubstituted aryloxycarbonyl (e.g. phenoxycarbonyl,p-methylphenoxycarbonyl, etc.),

substituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl,p-(p-methoxyphenylazo)benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, α-naphthylmethoxycarbonyl,p-biphenylisopropoxycarbonyl, etc.);

substituted or unsubstituted arenesulfonyl (e.g. benzenesulfonyl,p-toluenesulfonyl, etc.);

substituted or unsubstituted dialkylphosphoryl (e.g. dimethylphosphoryl,etc.);

substituted or unsubstituted diaralkylphosphoryl (e.g.0,0-dibenzylphosphoryl, etc.);

substituted or unsubstituted aryloxyalkanoyl (e.g. phenoxyacetyl,p-chlorophenoxyacetyl, 2-nitrophenoxyacetyl,2-methyl-2-(2-nitrophenoxy)propyonyl, etc.) or the like;

substituted or unsubstituted aryl such as phenyl, tolyl or the like;

substituted or unsubstituted aralkyl such as benzyl, diphenylmethyl,trityl, nitrobenzyl, or the like;

substituted or unsubstituted alkylidene (e.g. ethylene, isopropylidene,etc.) or the like;

substituted or unsubstituted aralkylidene such as benzylidene,2-hydroxybenzylidene, 2-hydroxy-5-chlorobenzylidene or the like;

substituted or unsubstituted arylthio such as phenylthio,nitrophenylthio, dinitrophenylthio, trichlorophenylthio or the like; and

substituted or unsubstituted aralkylthio such as tritylthio or the like.

(6) Esterified carboxy for R₃ ^(p), R_(c) ² and R₄ ^(q) :

The esterified carboxy and esterified carboxy moiety for R₃ ^(p), R_(c)² and R₄ ^(q) include a conventional esterified carboxy.

As suitable examples of the esterified carboxy, there may be exemplifiedthe same esters as illustrated above for explanation of protectedcarboxy for R², R^(p), R₁ ^(p) and R^(q), among which, as preferredexamples, there may be given an aliphatic hydrocarbon ester such asalkyl ester, i.e. alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl,etc.) and the like.

A pharmaceutically acceptable salt of the new peptides of the formula(I) may include a salt with an inorganic or organic base such as analkali metal salt (e.g. sodium salt, potassium salt, etc.), an alkalineearth metal salt (e.g. calcium salt, etc.), ammonium salt, ethanolaminesalt, triethylamine salt, dicyclohexylamine salt or the like, and anacid addition salt with organic or inorganic acid such as methanesulfonate, hydrochloride, sulfate, nitrate, phosphate or the like.

With regard to the formula (I) of the compound of this invention thefollowing is to be noted. That is, partial formula: ##STR10## in theformula (I) particularly means R¹, when n is an integer 0, ##STR11##when n is an integer 1, and ##STR12## when n is an integer 2. Further,partial formula ##STR13## in the formula (I) particularly means##STR14## when m is an integer 1, ##STR15## when m is an integer 2, and##STR16## when m is an integer 3.

The compound (I) of this invention including FR-900156 can be preparedby chemical synthetic methods and fermentation method, details of whichwill be apparent from the following description.

[1] Sythesis: ##STR17## [R¹, R_(b) ¹, R^(p), R², R^(q), R^(r), m and nare each as defined above provided that at least one of R^(p), R² andR^(q) is carboxy, or alternatively at least R_(a) ² is mono- ordi-carboxy lower alkyl or ar(carboxy)lower alkyl whose aryl moiety maybe substituted by hydroxy; and R_(c) ² and R₄ ^(q) are each hydrogen,carboxy, protected carboxy, esterified carboxy or a group of theformula: ##STR18## wherein R_(a) ² is mono- or di-carboxy (or protectedcarboxy) lower alkyl, esterified carboxy lower alkyl, ar(carboxy orprotected carboxy)lower alkyl whose aryl moiety may be substituted byhydroxy or ar(esterified carboxy)lower alkyl whose aryl moiety may besubstituted by hydroxy and R_(b) ² is as defined above, R₃ ^(p) ishydrogen, carboxy, protected carboxy or esterified carboxy provided thatat least one of R_(c) ², R₄ ^(q) and R₃ ^(p) is esterified carboxy, oralternatively at least R_(a) ² is mono- or di-esterified carboxy loweralkyl or ar(esterified carboxy)lower alkyl whose aryl moiety may besubstituted by hydroxy.]

[2] Fermentation

A strain belonging to the genus ##STR19## In the above formulae,

R_(c) ¹ is acyl,

R₁ ^(p) is hydrogen or protected carboxy,

R₂ ^(p) is hydrogen or carboxy,

R₁ ^(q) is hydrogen or protected carboxy,

R² and R₇ ^(q) are hydrogen, carboxy or a group of the formula:##STR20## wherein R_(a) ² is mono- or di-carboxy lower alkyl orar(carboxy)lower alkyl whose aryl moiety may be substituted by hydroxy,and R_(b) ² is as defined above,

R₁ ^(r) is amino protective group and

m,n, R¹, R², R^(p), R^(q), R^(r), R_(a) ² and R_(b) ² are each asdefined above.

Detailed explanation of processes for preparation of a new peptide ofthe formula (I) will be made in following.

[1] Synthesis

(1) Process 1: Peptide bond formation Compound (II)+Compound(III)→Compound (Ia)

This process relates to a method for preparing Compound (Ia) by reactingCompound (II) or its salt with a Compound (III) or its salt.

The reaction of this process can be conducted as follows.

That is, in one case, as the first step, the carboxy group of Compound(II) or its salt is usually activated in a conventional manner, forexample, in the form of its acid halide, azide, acid anhydride or amixed anhydride, activated ester, and the like, and is reacted with theCompound (III) to give Compound (Ia), and in the other case, theCompound (II) or its salt is reacted with the Compound (III) or its saltdirectly in the presence of a conventional condensing agent such asN,N-dicyclohexylcarbodiimide and the like. Among these activationmethods, preferred activation method for the carboxy group of theCompound (II) into its activated form and preferred condensing agent asmentioned above are selected according to kinds of the carboxyprotective group(s) of the Compound (II) and (III) and to the reactionconditions (e.g. the kinds of the reaction solvent, reaction temperatureand so on).

This reaction is preferably carried out in a solvent such as methylenechloride, chloroform, tetrahydrofuran, dioxane, ethyl acetate, methanol,ethanol, water or the like under ice-cooling to at ambient temperatureand the reaction in the presence of a condensing agent is usuallycarried out in an anhydrous, but not critical, conditions.

(2) Process 2: Selective deacylation Compound (Ia)→Compound (Ib)

This process relates to a method for preparing Compound (Ib) or its saltby removing selectively an acyl group for R_(c) ¹ of Compound (Ia) orits salt.

This process is applied to case that the acyl group for R_(c) ¹ revealsa different chemical property from that of the amino protective groupfor R₁ ^(r) against each kind of the removal methods and can selectivelybe removable by a method to be employed.

This reaction is carried out by conventional methods such as catalyticreduction method, liquidammoniaalkalimetal method, acid method, zincacid method, base method, hydrazine method and the like. Among thesemethods, preferred one is selected according to kind of the acyl groupfor R_(c) ¹ of Compound (Ia).

Each of the above methods is explained as follows.

(i) Catalytic reduction method:

This method is preferably applied to case that the acyl group for R_(c)¹ of Compound (Ia) are one which is removable by catalytic reduction. Aspreferred examples of such an acyl group for R_(c) ¹, there may beexemplified substituted or unsubstituted aralkoxycarbonyl (e.g.benzyloxycarbonyl, p-phenylazobenzyloxycarbonyl,p-(p'-methoxyphenylazo)benzyloxycarbonyl, p-nitrobenzyloxycarbonyl,etc.); substituted or unstubstituted alkenyl- or alkynyloxycarbonyl(e.g. allyloxycarbonyl, 1,1-dimethylproparglyloxycarbonyl, etc.);substituted or unsubstituted aryloxyalkanoyl (e.g. 2-nitrophenoxyacetyl,2-methyl-2-(2-nitrophenoxy)propionyl, etc.); and the like.

This catalytic reduction is carried out in a conventional manner, andsuitable catalysts to be used in catalytic reduction are conventionalones such as platinum catalyst (e.g. platinum plate, spongy platinum,platinum black, colloidal platinum, platinum oxide or platinum wire,etc.), palladium catalyst (e.g. spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium onbarium sulfate, palladium on barium carbonate, etc.), nickel catalyst(e.g. reduced nickel, nickel oxide, Raney nickel, etc.), cobalt catalyst(e.g. reduced cobalt, Raney cobalt, etc.), iron catalyst (e.g. reducediron, Raney iron, etc.), copper catalyst (e.g. reduced copper, Raneycopper, Ullman copper, etc.) or the like.

The reduction is usually carried out in a solvent. A suitable solvent tobe used may be, e.g. water, methanol, ethanol, propanol, ethyl acetate,tetrahydrofuran, dioxane, N,N-dimethylformamide, acetic acid, a mixtureof water and alcohol (e.g. methanol, ethanol, etc.) tetrahydrofuran,dioxane or ethyl acetate, and other conventional organic solvent or amixture thereof. Further, the reduction is preferably carried out in thepresence of an acid such as acetic acid or the like.

The reaction is preferably carried out under somewhat milder conditionssuch as cooling or warming.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand an amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive a Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.Further, in case that a protected carboxy group for R^(p) is, forexample, substituted or unsubstituted aralkyl ester type one (e.g.benzyl ester p-nitrobenzyl ester, p-chlorobenzyl ester,p-phenylazobenzyl ester, etc.), such a protective group also issimultaneously removed in this process to give a Compound (Ib) whereinR^(p) is carboxy.

(ii) Acid method:

(ii)-1 Method of use of trifluoroacetic acid or formic acid:

This method is preferably applied to case that the acyl group for R_(c)¹ is one which is removable by treating with trifluoro-acetic acid orformic acid. Preferred examples of such an acyl group may be exemplifiedby a group such as branched- or alicyclicoxycarbonyl, (e.g.t-butoxycarbonyl, t-pentoxycarbonyl, t-amyloxycarbonyl,adamantyloxycarbonyl, isobornyloxycarbonyl, etc.); substituted orunsubstituted aralkoxycarbonyl (e.g. p-methoxybenzyloxycarbonyl, etc.).

This reaction is conventionally carried out in a solvent such asmethylene chloride, chloroform, acetic acid, water and the like in thepresence of trifluoroacetic acid or formic acid, and anisole ispreferably added thereto.

Trifluoroacetic acid and formic acid are also used as the solvent.

This reaction is usually carried out under ice-cooling to at ambienttemperature.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand an amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive a Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.Further, in case that a protected carboxy group for R^(p) is, forexample, a branched alkyl ester (e.g. t-butyl ester, etc.), orsubstituted or unsubstituted aralkyl ester (e.g. diphenylmethyl ester,p-methoxybenzyl ester, etc.), such a protective group also issimultaneously removed to give a Compound (Ib) wherein R^(p) is carboxy.

(ii)-2 Method of use of hydrochloric acid or p-toluenesulfonic acid:

This method is preferably applied to case that an acyl group for R_(c) ¹is one which is removed by treating with hydrochloric acid orp-toluenesulfonic acid.

Preferred examples of such an acyl group may be exemplified by e.g.substituted or unsubstituted branched alkoxycarbonyl (e.g.t-butoxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, etc.) and thelike in addition to one as illustrated in the above (ii)-1.

This reaction is carried out in a solvent such as ethyl acetate,methylene chloride, chloroform, tetrahydrofuran and the like in thepresence of an inorganic or organic strong acid such as hydrochloricacid, p-toluenesulfonic acid or the like, and anisole is preferablyadded thereto.

This reaction is preferably carried out under ice-cooling to at ambienttemperature.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand an amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive a Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.Further, in case that a protected carboxy group for R^(p) is, forexample, a branched alkyl ester (e.g. t-butyl ester etc.) or substitutedor unsubstituted aralkyl ester (e.g. diphenylmethyl ester,p-methoxybenzyl ester, etc.), such a protective group also issimultaneously removed to give a Compound (Ib) wherein R^(p) is carboxy.

(ii)-3 Method of use of hydrogen bromide:

This method is preferably applied to case that an acyl group for R_(c) ¹is one which is removable by treating with hydrogen bromide.

Preferred examples of such an acyl group may be exemplified bysubstituted or unsubstituted aralkoxycarbonyl (e.g. benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-tolyloxycarbonyl,p-phenylazobenzyloxycarbonyl, α-naphthylmethoxycarbonyl, etc.) and analkoxycarbonyl (e.g. isopropoxycarbonyl, etc.) in addition to one asillustrated in the above (ii)-1 and (ii)-2.

This reaction is usually carried out in a solvent such as ethyl acetate,acetic acid, trifluoroacetic acid or the like in the presence ofhydrogen bromide.

This reaction is preferably carried out under ice-cooling to at ambienttemperature.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand an amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.Further, in case that a protected carboxy group for R^(p) is, forexample, a branched alkyl ester or (e.g. t-butyl ester, etc.),substituted or unsubstituted aralkyl ester (e.g. diphenylmethyl ester,p-methoxybenzyl ester, etc.), such a protective group is simultaneouslyremoved to give a Compound (Ib) wherein R^(p) is carboxy.

(iii) Liquid-ammonia-alkali metal method:

This method is preferably applied to case that the acyl group for R_(c)¹ is one which is removable by treating with liquid ammonia-alkalimetal. As preferred examples of such an acyl group, there may beexemplified substituted or unsubstituted aralkoxycarbonyl (e.g.benzyloxycarbonyl, etc.), substituted or unsubstituted aryloxycarbonyl(e.g. phenoxycarbonyl, p-methylphenoxycarbonyl, etc.), an arenesulfonyl(e.g. benzenesulfonyl, p-toluenesulfonyl, etc.) and the like.

This reaction is usually carried out by dissolving Compound (Ia) intoliquid ammonia and then alkali metal is added thereto.

This reaction is preferably carried out at a lower temperature, e.g. at-78° C. to at boiling point of liquid ammonia.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive a Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.

(iv) Hydrazine method:

This method is preferably applied to case that the acyl group for R_(c)¹ is one which is removable by treating with a hydrazine compound or anamine compound. As preferred examples of such an acyl group, there maybe exemplified phthaloyl, formyl, acetoacetyl, etc.

Preferred examples of hydrazine compound are exemplified by hydrazine,methylhydrazine, phenylhydrazine and the like and those of aminecompound are exemplified by hydroxylamine, dialkylaminoalkylamine (e.g.N,N-dimethylaminopropylamine, etc.) and the like.

This reaction is usually carried out by treating Compound (Ia) with thehydrazine compound or amine compound in a solvent such as water, alcohol(e.g. methanol, ethanol, etc.) tetrahydrofuran, dioxane or the like atambient temperature to under reflux.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand an amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive a Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.

(v) Zinc-acid method:

This method is preferably applied to case that the acyl group for R_(c)¹ is one which is removable by treating with zinc acid. As preferredexamples of such an acyl group, there may be exemplifiedtrichloroethoxycarbonyl, 4-piperidyloxycarbonyl,1-methyl-1-(4-pyridyl)ethoxycarbonyl and the like.

This method is carried out by treating Compound (Ia) with zinc in thepresence of a weak acid such as formic acid, acetic acid and the like.The reaction may be carried out in a solvent such as methylene chloride,chloroform, tetrahydrofuran, ethyl acetate, alcohol (e.g. methanol,ethanol, etc.), dimethylformamide and the like, and in this case a weakacid as mentioned above is added to such a solvent. The reaction isusually carried out at -10° C. to ambient temperature.

In this reaction, in case that R^(q) is a group of the formula:--CONHNHY and an amino protective group for Y is the same as the acylfor R_(c) ¹, then such an amino protective group also is simultaneouslyremoved to give a Compound (Ib) wherein R^(q) is a group of the formula:--CONHNH₂. Further, in case that a protected carboxy group for R^(p) ofCompound (Ia) is, for example, a halo-alkyl ester type of group (e.g.trichloroethyl, etc.), such a carboxy protective group also issimultaneously removed to give a Compound (Ib) wherein R^(p) is carboxy.

(vi) Base method:

This method is preferably applied to case that the acyl group for R_(c)¹ is one which is removable by treating with a base. As preferredexamples of such an acyl group, there may be exemplified haloalkanoyl(e.g. trifluoroacetyl, etc.), substituted or unsubstitutedalkoxycarbonyl (e.g. 2-(p-toluenesulfonyl)ethoxycarbonyl,2-(p-tolylthio)ethoxycarbonyl, etc.), substituted or unsubstitutedaryloxycarbonyl (e.g. 2-nitrophenoxycarbonyl, etc.) and the like.

This method is carried out in the presence of a base under ice-coolingto at ambient temperature.

Suitable base is an inorganic base such as alkali metal hydroxide oralkaline earth metal hydroxide, or the corresponding carbonate orbicarbonate (e.g. sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate, lithium carbonate, sodium bicarbonate,calcium hydroxide, magnesium hydroxide, etc.), ammonium hydroxide or thelike; an organic base such as an alkoxide or phenoxide of the abovemetal (e.g. sodium ethoxide, sodium methoxide, lithium phenoxide, etc.),an amine such as mono-, di- or tri-alkylamine (e.g. methylamine,ethylamine, propylamine, isopropylamine, butylamine,N,N-dimethyl-1,3-propanediamine, trimethylamine, triethylamine, etc.),unsubstituted, mono- or disubstituted arylamine (e.g. aniline,N-methylaniline, N,N-dimethylaniline, etc.), a heterocyclic base (e.g.pyrrolidine, morpholine, N-methylmorpholine, N-methylpiperidine,N,N-dimethylpiperazine, pyridine, etc.) or the like; a basic ionexchange resin and the like.

This method is preferably conducted under somewhat milder conditionssuch as coolind or warming and usually in any solvent which does nothave an adverse influence on the reaction, e.g. water, a hydrophilicsolvent such as alcohol (e.g. methanol, ethanol, propanol, etc.),N,N-dimethylformamide, tetrahydrofuran, dioxane, dimethylsulfoxide, etc.or a mixture thereof. In case that the above-mentioned bases are inliquid, they can also be used as a solvent.

In this method, in case that R^(q) is a group of the formula: --CONHNHYand an amino protective group for Y is the same as the acyl for R_(c) ¹,then such an amino protective group also is simultaneously removed togive a Compound (Ib) wherein R^(q) is a group of the formula: --CONHNH₂.Further, in case that a protected carboxy group for R^(p) of Compound(Ia) is, for example, an alkyl ester type of group (e.g. methyl ester,ethyl ester, etc.), an aralkyl ester type group (e.g. benzyl ester,etc.), such a protective group also is simultaneously removed to give aCompound (Ib) wherein R^(p) is carboxy.

(3) Process 3: ##STR21##

This process relates to a method for preparing Compound (Ia) by reactingCompound (Ib) with an acylating agent.

The acylating agent to be used in this reaction includes an organic acid(R_(c) ¹ --OH wherein R_(c) ¹ is acyl group) such as monobasic ordibasic organic carboxylic acid, an organic carbonic acid or an organiccarbamic acid and the corresponding thio acid or imidic acid; and anorganic sulfonic acid, and more particularly, aliphatic, aromatic orheterocyclic carboxylic acid, and the corresponding carbonic, carbamic,thiocarboxylic, thiocarbonic, thiocarbamic, carboximidic, carbamimidicacid, and sulfonic acid; their reactive derivatives; and also includesan isocyanate (e.g. potassium-, alkyl- or aryl-isocyanate),isothiocyanate (e.g. alkyl isothiocyanate) and an isothiourea (e.g.ethyl isothiourea). Suitable examples of these organic acid (R_(c) ¹--OH wherein R_(c) ¹ is acyl group) are the corresponding organic acidto those comprising the acyl group is exemplified hereinabove in detailsin the descriptions of suitable examples of acyl groups for R¹ and R_(c)¹ of the compound (I).

Said organic acid as an acylating agent can be used in the form of anactivated organic acid, i.e. as a reactive derivative of the acid. Assuch reactive derivatives of said organic acids, there may beexemplified an acid halide, an acid azide, an acid anhydride, anactivated amide, an activated ester, etc., and additionally isocyanateand isothiocyanate can preferably be used as reactive derivative ofcarbamic and thiocarbamic acids, respectively. Preferred examples ofsuch reactive derivatives are illustrated by:

an acid halide (e.g. acid chloride, acid bromide etc.);

an acid azide;

an acid anhydride including a mixed acid anhydride with an acid such asdialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid,dibenzylphosphoric acid, halogenated phosphoric acid, dialkylphosphorousacid, sulfurous acid, thiosulfuric acid, sulfuric acid,monoalkylcarbonic acid, aliphatic carboxylic acid (e.g. acetic acid,pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid,trichloroacetic acid, etc.), aromatic carboxylic acid (e.g. benzoicacid, etc.) or the like, and symmetrical acid anhydride;

an activated amide with pyrazole, imidazole, 4-substituted imidazole,dimethylpyrazole, triazole or tetrazole; and

an activated ester such as substituted or unsubstituted alkylthio ester(e.g. methythio ester, carboxymethyl thioester, etc.), substituted orunsubstituted aryl thioester (e.g. phenyl thioester, p-nitrophenylthioester, p-cresyl thioesther, etc.), heterocyclic ester (e.g. pyranylester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, etc.) orester with N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone,N-hydroxysuccinimide, N-hydroxyphthalimide or1-hydroxy-6-chlorobenzotriazole, or the like.

The above reactive derivative is selected according to the kind of theacid to be used.

In the reaction, when free acid is used as an acylating agent, theacylation reaction may preferably be conducted in the presence of acondensing agent such as carbodiimidic compound (e.g.N,N'-dicyclohexylcarbodiimide,N-cyclohexyl-N'-morpholinoethylcarbodiimide,N-cyclohexyl-N'-(4-diethylaminocyclohexyl)carbodiimide,N,N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide,N-ethyl-N'-(3-dimethylamino-propyl)carbodiimide, etc.),N,N'-carbonyldi(2-methylimidazole),pentamethyleneketene-N-cyclohexylimine,diphenylketene-N-cyclohexylimine, alkoxyacetylene,1-alkoxy-1-chloroethylene, trialkyl phosphite, ethyl polyphosphate,isopropyl polyphosphate, phosphorus compound (e.g. phosphorusoxychloride, phosphorus trichloride, etc.), thionyl chloride, oxalylchloride, 2-ethyl-7-hydroxybenzisoxazolium salt,2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide,(chloromethylene)-dimethylammoniumchloride,2,2,4,4,6,6,-hexachloro-1,3,5,2,4,6-triazatriphosphorine,1-benzenesulphonyloxy-6-chloro-1H-benzotriazole, p-toluenesulfonylchloride, isopropoxybenzenesulfoxyl chloride or the like; or a mixedcondensing agent such as a mixture of triphenylphosphine and a carbontetrahalide (e.g. carbon tetrachloride, carbon tetrabromide, etc.), acomplex of N,N-dimethylformamide with phosphoryl chloride, phosgene orthionyl chloride, etc., or the like.

The reaction is usually conducted in a solvent such as water, alcohol(e.g. methanol, ethanol, propanol, etc.), acetone, ethyl ether, dioxane,acetonitrile, ethylacetate, N,N-dimethylformamide, dimethylsulfoxide,tetrahydrofuran, dichloromethane, chloroform, etc. or pyridine,N-methylmorpholine, N-methylpyrrolidine or other conventional solvents,or a mixture thereof.

The reaction can also be conducted preferably in the presence of anorganic or inorganic base such as alkali metal (e.g. sodium, potassium,etc.), alkaline earth metal (e.g. calcium, etc.), alkali or alkalineearth metal hydride (e.g. sodium hydride, calcium hydride, etc.), alkalior alkaline earth metal hydroxide (e.g. sodium hydroxide, potassiumhydroxide, calcium hydroxide, etc.), alkali or alkaline earth metalcarbonate or bicarbonate (e.g. sodium carbonate, potassium carbonate,sodium bicarbonate, lithium carbonate, etc.), alkali or alkaline earthmetal alkoxide (e.g. sodium ethoxide, lithium methoxide, magnesiummethoxide, etc.), trialkylamine (e.g. triethylamine, etc.), pyridine,bicyclodiaza compound (e.g. 1,5-diazabicyclo[3,4,0]nonene-5,1,5-diazabicyclo[5,4,0]undecene-5, etc.) or the like. Among said base, aliquid one can also be used as a solvent.

There is no limination to this reaction temperature, and this reactionmay preferably be conducted within the range of cooling to ambienttemperature.

(4) Process 4: Elimination of protective groups Compound (Ic)→Compound(Id)

This process relates to a method for preparing Compound (Id) bysubjecting Compound (Ic) to removal reaction of protective groups ofprotected carboxy groups for R^(p) and R^(q) and or amino protectivegroup for R^(r), details of which are explained as follows:

Process 4-1: Elimination of an amino protective group for R^(r)

The elimination of an amino protective group for R₁ ^(r) is carried outsubstantially in the same manner as that of Process 2, and accordinglythe detailed explanation for Process 2 as made hereinabove is to bereferred to.

Process 4-2: Removal of carboxy protective group of protected carboxyfor R^(p) and R^(q)

The reaction for removal of protective group of the protected carboxygroup is carried out by a conventional method such as hydrolysis andreduction or the like, details of which are explained in the following.

(i) For hydrolysis which refers to the same meaning as solvolysisincluding, for example, acidolysis, alcoholysis, aminolysis,hydroxinolysis, etc.:

Hydrolysis is preferably carried out in the presence of an acid or base.

Suitable acid includes an inorganic acid (e.g. hydrochloric acid,hydrobromic acid, sulfuric acid, etc.), an organic acid (e.g. formicacid, acetic acid, trifluoroacetic acid, propionic acid, benzenesulfonicacid, p-toluenesulfonic acid, etc.), an acidic ion-exchange resin andthe like. Suitable base includes an inorganic base such as alkali oralkaline earth metal hydroxide or the corresponding carbonate orbicarbonate (e.g. sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate, lithium carbonate, sodium bicarbonate,calcium hydroxide, magnesium hydroxide, etc.), ammonium hydroxide or thelike; an organic base such as an alkoxide or phenoxide of the abovemetal (e.g. sodium ethoxide, sodium methoxide, lithium phenoxide, etc.),an amine such as mono-, di- or tri-alkylamine (e.g. methylamine,ethylamine, propylamine, isopropylamine, butylamine,N,N-dimethyl-1,3-propanediamine, trimethylamine, triethylamine, etc.),unsubstituted, mono- or disubstituted arylamine (e.g. aniline,N-methylaniline, N,N-dimethylaniline, etc.), a heterocyclic base (e.g.pyrrolidine, morpholine, N-methylmorpholine, N-methylpiperidine,N,N-dimethylpiperazine, pyridine, etc.), hydrazines (e.g. hydrazine,methylhydrazine, ethylhydrazine, etc.) or the like; a basic ion-exchangeresin and the like.

The hydrolysis is preferably conducted under somewhat milder conditionssuch as cooling or warming and usually in a solvent which does not haveadverse influence to the reaction, e.g. water, a hydrophilic solventsuch as alcohol (e.g. methanol, ethanol, propanol, etc.), acetone,N,N-dimethylformamide, tetrahydrofuran, dioxane, dimethylsulfoxide, etc.or a mixture thereof, and other hydrophobic solvent such as benzenediethylether, etc. may also be used as a solvent. A liquidabovementioned acid or base can also be used as solvent.

(ii) For reduction:

Reduction, including chemical reduction and catalytic reduction, iscarried out in a conventional manner.

Suitable reducing agents to be used in chemical reduction are a metal(e.g. tin, zinc, iron, etc.), or a combination of such metal and/ormetallic compound (e.g. chromium chloride, chromium acetate, etc.) andan organic or inorganic acid (e.g. formic acid, acetic acid, propionicacid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid,hydrobromic acid, etc.).

Suitable catalysts to be used in catalytic reduction are conventionalones such as platinum catalysts (e.g. platinum plate, spongy platinumplatinum black, colloidal platinum, platinum oxide or platinum wire,etc.), palladium catalysts (e.g. spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium anbarium sulfate, palladium on barium carbonate, etc.), nickel catalysts(e.g. reduced nickel, nickel oxide, Rabey nickel, etc.), cobaltcatalysts (e.g. reduced cobalt, Raney cobalt, etc.), iron catalysts(e.g. reduced iron, Raney iron, etc.), copper catalysts (e.g. reducedcopper, Raney copper, Ullman copper, etc.) or the like.

The reduction is usually carried out in a solvent. A suitable solvent tobe used may be, e.g. water, alcohol (e.g. methanol, ethanol, propanol,etc.) and other conventional organic solvent or a mixture thereof.Additionally, the afore-mentioned liquid acids to be used in chemicalreduction can also be used as solvent. Further, a suitable solvent to beused in catalytic reduction may be, e.g., the above-mentioned solvent,and other conventional solvent, such as diethyl ether, dioxane,tetrahydrofuran, etc. or a mixture thereof.

The reaction is preferably carried out under somewhat milder conditionssuch as cooling or warming.

Among these methods for removal of protective groups, preferred one andappropriate combination methods are to be selected according to kinds ofthe protective groups of carboxy group and amino protective group to beremoved off.

It is noted that this process includes the following cases of removal ofprotective groups and amino protective group, that is, one case that allof the carboxy protective groups for R^(p) and R^(q) and the aminoprotective group for R^(r) in the Compound (Ic) are simultaneouslyremoved by a method to be employed to the reaction, and the other casethat the carboxy protective groups and the amino protective group aresequentially and stepwise removed by a method which is appropriatelyselected according to the kinds of the protective group to be removed.

Process 4-3: Removal of hydrazino group

A protected carbazoyl of the formula: --CONHNHY wherein Y is an aminoprotective group can be removed by subjecting the Compound (Ic) at firstto the reaction of Process 4-1 for eliminating amino protective group(i.e. Y) to give --CO.NHNH₂ group and then subjecting the reactionproduct to the reaction of this step to give --COOH group, andparticular of this reaction step is as follows.

The reaction of this step is carried out in a conventional manner bytreating the Compounds (Ic) with a conventional oxidizing agent which iscapable of oxidizing a group of the formula: --CONHNH₂ to form into agroup of the formula: --COOH and accordingly preferred example of suchan oxidizing agents may be halogen such as iodine, bromine etc.,perhalogenic acid such as periodic acid or its salt (e.g. sodium salt,potassium salt, etc.), perchloric acid, etc., N-haloimide such asN-bromosuccinimide, etc., lead tetraacetate, hydrogen peroxide or itssalt (e.g. nickel peroxide, etc.), metal oxide such as mercuric oxide,manganese dioxide, nickel peroxide, etc., cupric compound (e.g. cupricacetate, cupric sulfate, etc.) and the like.

This reaction is usually carried out in a solvent such as water, aceticacid, methanol, ethanol, tetrahydrofuran, dioxane and the like and amixture thereof, which should be appropriately selected in accordancewith the kind of oxidizing agent to be used.

This reaction is usually carried out under icecooling to at ambienttemperature, or under reflux.

As to Process 4 for Elimination of protective groups (i.e. Process 4-1,Process 4-2 and Process 4-3), it is to be noted that, in case that acylfor R¹ has one or more protective groups for hydroxy, amino and (or)carboxy, such protective groups also may be simultaneously removed andsuch a case is included within the scope of this process.

(5) Process 5: Peptide bond formation 2 Compound (Ig)→Compound (Ih)

This process relates to a method for preparing Compound (Ih) by reactingCompound (Ig) or its salt with a compound of the formula: ##STR22## orits salt (wherein R_(a) ² and R_(b) ² are each as defined above).

This reaction is carried out in substantially the same manner as that ofProcess 1, and accordingly the detailed explanation for Process 1 asmade hereinabove is to be referred thereto.

(6) Process 6: Aminoprotection Compound (Ij)→Compound (Ia)

This process relates to a method for preparing Compound (Ia) by reactingCompound (Ij) with an amino protecting agent.

The amino protecting agent to be used in this reaction is the same asone illustrated in Process 6^(s) explained hereinafter.

The reaction is carried out substantially in the same manner as that ofProcess 3.

(7) Process 7: Esterification

This process relates to a method for preparing Compound (Il) by reactingCompound (Ik) with an esterifying agent.

An esterifying agent to be used in this reaction may include aconventional one such as alcohol (e.g. methanol, ethanol, propanol,butanol, benzylalcohol etc.) or its reactive equivalent (e.g. halide,sulfate, aliphatic or aromatic sulfonate or the corresponding diazocompound, etc.) and the like.

This reaction is carried out in a conventional manner and in case ofusing alcohol as an esterifying agent the reaction is usually carriedout in the presence of an acid such hydrochloric acid, sulfuric acid,methanesulfonic acid or the like, and in case of using alkyl halide asan esterifying agent the reaction is usually carried out in the presenceof a base as illustrated in the aforementioned Process 2.

(2) Fermentation: A strain of Streptomyces→Compound (Ie)

This compound (Ie) (hereinafter referred to as FR-900156 substance) canbe produced by fermentation of a FR-900156 substance producing strainbelonging to the genus Streptomyces in a nutrient medium, details ofwhich are explained in the following.

The microorganism which can be used for the production of the FR-900156substance is a strain belonging to the genus Streptomyces, among which astrain of Streptomyces olivaceogriseus and Streptomyces violaceus havebeen newly isolated from a soil sample as a suitable strain of aFR-900156 substance-producing strain belonging to the genusStreptomyces.

It is to be understood that, for the production of the FR-900156substance, this invention is not limited to the use of the particularorganism as described herein, which is given for illustrative purposeonly. This invention also includes the use of any mutants which arecapable of producing the FR-900156 substance, including natural mutantswhich are produced by natural mutation of the organisms as well asartificial mutants which can be produced from the described organisms byconventional means, such as X-rays, ultra-violet radiation, nitrogenmustard oils and the like.

I. Re. Streptomyces olivaceogriseus nov. sp. C-353:

Streptomyces olivaceogriseus nov. sp. C-353 has been isolated from asoil sample collected at Kochi Prefecture, Japan and deposited with andadded to a permanent stock culture collection of the America TypeCulture Collection under the number ATCC 31427.

Streptomyces olivaceogriseus nov. sp. C-353 (ATCC 31427) has thefollowing morphological, cultural and physiological characteristics.

(1) Morphological characteristics:

Microscopic observations were made on cultures which were grown from 10to 14 days on sucrose-nitrate agar, glycerin-asparagine agar, yeastmaltextract agar, oatmeal agar, starch-inorganic salts agar and Bennettagar. Sporophore morphology was observed on undisturbed plates cultures.

1. Type of branching of spore-forming hyphae: Monopodial branching

2. Form of spore-forming hyphae:

Retinaculiaperti

(closed spirals, loops)

3. Numbers of spores: 5-20 spores

4. Surface appearance and size of spores:

Smooth

0.6-1.2×1.0-1.9 micron

5. Existence of zoospores: Not observed

6. Existence of sporangium: Not observed

7. Formation of spores: At aerial mycelium

8. Fragmentation of substrate mycelium: Not observed

(2) Cultural characteristics:

The following observations were made on slant cultures which were grownon various media at 30° C. for 10-14 days.

    ______________________________________                                                   Aerial mass Reverse side Soluble                                   Medium     color       of colony    Pigment                                   ______________________________________                                        Sucrose-nitrate                                                                          none or     pale yellow, none                                      agar       very thin,  small colonies                                                    powdery                                                            Glucose-aspara-                                                                          light gray to                                                                             pale yellow to                                                                             none                                      gine agar  greenish gray,                                                                            pale yellowish                                                    powdery     brown, small                                                                  colonies                                               Glycerin-aspara-                                                                         thin, powdery                                                                             pale yellowish                                                                             none or                                   gine agar  light gray  brown,       trace                                                            small colonies                                         Starch-inorganic                                                                         olive gray, grayish yellow                                                                             none                                      salts agar powdery     brown,                                                                        small colonies                                         Tyrosine agar                                                                            light olive yellowish brown,                                                                           light                                                gray, thin  small colonies                                                                             brown                                                powdery                                                            Nutrient agar                                                                            none        pale yellow, none                                                             flat                                                   Yeast-malt greenish gray,                                                                            yellowish brown,                                                                           none                                      extract agar                                                                             powdery     wrinkled                                                                      colonies                                               Oatmeal agar                                                                             light gray to                                                                             colorless,   none                                                 greenish gray,                                                                            small colonies                                                    powdery                                                            Peptone-yeast                                                                            none        colorless to light                                     iron agar              pale yellow, brown                                                            slightly wrinkled                                      Glucose-peptone                                                                          white,      colorless to pale                                                                          brown                                     gelatin stab                                                                             thin powdery                                                                              yellow, wrinkled                                                              colonies                                               Milk       white,      colorless    none                                                 very thin   surface ring                                                      powdery     growth                                                 ______________________________________                                    

(3) Biological and physiological properties:

1. Temperature requirements (on Bennett agar slants) growth from 15° C.to 40° C. optimum 28° C.

2. Hydrolysis of startch (on starch-inorganic salts agar) hydrolyzedweakly

3. Liquefaction of gelatin (on glucose-peptone gelatin stab) negative

4. Action on milk

no coagulation

no peptonization

5. Production of melanin (on tyrosine agar, peptone-yeast iron agar andtryptone-yeast broth) positive

6. Utilization of various carbon compounds (on Pridham-Gottlieb basalagar medium)

L-Arabinose: -

D-Xylose: ±

D-Glucose: +

D-Fructose: +

D-Galactose: +

Sucrose: +

Glycerin: +

Inositol: +

Lactose: +

L-Rhamnose: -

Maltose: +

Raffinose: -

D-Mannitol: +

D-Mannose: +

Salicin: -

Symbols:

+, good utilization; ±, doubtful utilization;

-, no utilization

7. Cell wall pattern I (LL-diaminopimelic acid)

As a result of looking up the strain possessing the characteristicsmentioned above by referring to the literature, namely "Bergey's Manualof Determinative Bacteriology" eighth edition (1975), and "TheInternational Streptomyces Project Reports" written by E. B. Shirlingand D. Gottlieb Cf. International Journal of Systematic BacteriologyVol. 18, pages 69 and 279 (1968), Vol. 19, pages 391 (1969) and Vol. 22,pages 265 (1972), Streptomyces eurythermus and Streptomyces galbus(Okami) have been detected as species having relatively analogouscharacteristics to those of the strain ATCC 31427.

The strain ATCC 31427, however, is different from these analogousspecies in the following:

Streptomyces eurythermus (Okami):

A strain of the species can assimilate arabinose and can not assimilateinositol. Assimilation of rhamnose and raffinose by a strain of thespecies is indefinite. Loops are not formed. Straights or flexousmycelium are sometimes observed.

Streptomyces galbus:

Open-spirals are generally formed. A strain of the species produces asoluble yellow--yellow green pigment, and can not assimilate sucrose.

In view of the result of the observation, the strain ATCC 31427 can bejudged to be a new species belonging to the genus Streptomyces and thishas been designated as Streptomyces olivaceogriseus nov. sp. C-353.

II. Re. Streptomyces violaceus No.6724

Streptomyces violaceus No.6724 was isolated from a soil sample collectedat Ishigaki island, at Okinawa Prefecture, Japan, and deposited with andadded to a permanent stock culture collection of the American TypeCulture Collection under the number ATCC 31481.

Streptomyces violaceus No.6724 has following morphological, cultural andphysiological characteristics.

(1) Morphological characteristics

Microscopic observations were made on cultures which were grown onsucrose-nitrate agar, glycerin-asparagine agar, starch-inorganic saltsagar, yeast-malt extract agar and oatmeal agar at 30° C. for 10 to 14days.

1. Type of branching of spore-forming hyphae: Monopodial branching

2. Form of spore-forming hyphae: Spirales

3. Numbers of spores: 10-50

4. Surface appearance and size of spore:

Spiny

0.3-0.7×0.6-1.1μ

5. Existence of zoospore and sporangium: Not observed

6. Formation of spores: At aerial mycelium

7. Fragmentation of substrate mycelium: Not observed.

(2) Cultural characteristics

The following observations were made on cultures which were grown onvarious madia at 30° C. for 10 to 14 days.

    ______________________________________                                                   Aerial mass Reverse side Soluble                                   Medium     color       of colony    Pigment                                   ______________________________________                                        Sucrose-nitrate                                                                          purplish white,                                                                           small colonies                                                                             purple                                    agar       powdery                                                            Glucose-aspara-                                                                          purplish white,                                                                           yellowish red,                                                                             pink                                      gine agar  powdery     small colonies                                         Glycerin-aspara-                                                                         pinkish white-                                                                            Yellowish red,                                                                             pink-                                     gine agar  pink, powdery                                                                             small colonies,                                                                            red                                                              slightly wrinkled                                      Starch-inorganic                                                                         whitish red,                                                                              yellowish red,                                                                             red                                       salts agar short cottony                                                                             slightly wrinkled                                      Tyrosine agar                                                                            none        red, wrinkled                                                                              none or                                                          colonies     trace                                     Nutrient agar                                                                            none or very                                                                              colorless-purple,                                                                          reddish                                              thin powdery                                                                              flat         purple                                    Yeast-malt pink-purplish                                                                             reddish brown-                                                                             reddish                                   extract agar                                                                             pink,       purplish brown,                                                                            purple                                               short cottony                                                                             wrinkled colonies                                      Oatmeal agar                                                                             pink-purplish                                                                             colorless-   pink-                                                pink, powdery                                                                             purplish pink,                                                                             purple                                                           small colonies                                         Peptone-yeast                                                                            none        colorless,   brown                                     iron agar              wrinkled colonies                                      Glucose-peptone                                                                          white, thin red, wrinkled                                                                              faint                                     gelatin stab                                                                             powdery     colonies     brown                                     Milk       thin powdery                                                                              red growth on                                                                              reddish                                                          surface      yellow                                    ______________________________________                                    

Reverse mycelium pigment is pH indicator, changing from red to violet(purple) with addition of 0.05N NaOH or from violet to red (pink) withaddition of 0.05N HCl. Soluble pigment is also pH sensitive, showing thesame changes noted for reverse mycelium pigment.

(3) Biological and physiological properties

1. Temperature requirements (on Bennett agar slants) growth from 15° C.to 40° C. (optimum 28° C.)

2. Liquefaction of gelatin (on glucose-peptone gelatin stab) negative

3. Hydrolysis of starch (on starch-inorganic salts agar) positive

4. Action of milk: coagulation, no peptonization

5. Production of melanoid pigment (on tyrosine agar, peptone-yeast ironagar and tryptone-yeast extract broth)

positive

very weak or not on tyrosine agar

6. Utilization of various carbon compounds (on Pridham-Gottlieb basalagar medium)

L-Arabinose: +

D-Xylose: +

L-Rhamnose: +

D-Glucose: +

D-Fructose: +

D-Mannose: +

D-Galactose: +

Sucrose: +

Lactose: +

Maltose: +

Raffinopse: +

Inulin: ±

Cellulose: -

Chitin: -

Glycerin: +

D-Mannitol: +

Salicin: +

Inositol: +

Na-Acetate: -

Na-Citrate: +

Na-Succinate: +

Symbols:

+, good utilization

±, doubtful utilization

-, no utilization.

The FR-900156 substance of this invention is produced when the FR-900156substance-producing strain belonging to the genus Streptomyces (e.g.Streptomyces olivaceogriseus nov, sp-C-353 and Streptomyces violaceus,etc.) is grown in a nutrient medium containing sources of assimilablecarbon and nitrogen under aerobic conditions (e.g. shaking culture,submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, fructose, glycerin, starch and the like. Other sourceswhich may be included are lactose, arabinose, xylose, dextrin, molassesand the like.

The preferred sources of nitrogen are yeast extract, peptone, glutenmeal, cottonseed meal, soybean meal, corn steep liquor, dried yeast,wheat germ, etc., as well as inorganic and organic nitrogen compoundssuch as ammonium salts (e.g. ammonium nitrate, ammonium sulphate,ammonium phosphate, etc.), urea, amino acid and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form because less purematerials, which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use. Whendesired, there may be added to the medium mineral salts such as calciumcarbonate, sodium or potassium phosphate, sodium or potassium chloride,magnesium salts, copper slats and the like. If necessary, especiallywhen the culture medium foams seriously a defoaming agent, such asliquid paraffin, fatty oil, plant oil, mineral oil or silicone may beadded.

As in the case of the preferred methods used for the production of otherantibiotics in massive amounts, submerged aerobic cultural conditionsare preferred for the production of the FR-900156 substance in massiveamounts. For the production in small amounts, a skaking or surfaceculture in a flask or bottle is employed. Furthermore, when the growthis carried out in large tanks, it is preferable to use the vegetativeform of the organism for inoculation in the production tanks in order toavoid growth lag in the process of production of the FR-900156substance. Accordingly, it is desirable first to produce a vegetativeinoculum of the organism by inoculating a relatively small quantity ofculture medium with spores or mycelia of the organism and culture themand then to transfer the cultured vegetative inoculum aseptically tolarge tanks. The medium, in which the vegerative inoculum is produced,is substantially the same as or different from the medium utilized forthe production of the FR-900156 substance.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 20°C. and 40° C., preferably 30° C., for a period of about 50 hours to 100hours.

The FR-900156 substance can be recovered from the culture medium byconventional means which are commonly used for the recovery of otherknown antibiotics.

In general, most of the FR-900156 substance produced are found in thecultured broth, and accordingly the FR-900156 substance can be separatedfrom the filtrate, which is obtained by filtering of centrifuging theculture broth, by a conventional method such as concentration underreduced pressure, lyophilization, pH adjustment, treatment with a resin(e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.),treatment with an adsorbent (e.g. activated charcoal, silicic acid,silica gel, cellulose, alumina, etc.), crystallization,recrystallization and the like.

The FR-900156 substance thus produced in the culture broth can beisolated in the free form, i.e., FR-900156 substance per se and when thesolution or its concentrate containing the FR-900156 substance istreated with a base, i.e. with an inorganic base such as an alkali metalcompound (e.g. sodium hydroxide, sodium carbonate, sodium bicarbonate,potassium hydroxide, etc.), an alkaline earth metal compound (e.g.calcium hydroxide, magnesium hydroxide, etc.), ammonia and the like,with an organic base (e.g. ethanolamine, triethylamine,dicyclohexylamine, etc.); or with an acid i.e. with an inorganic acid(e.g. hydrochloric acid, sulfuric acid, phosphoric acid, etc.); or withan organic acid (e.g. formic acid, acetic acid, p-toluenesulfonic acid,citric acid, tartaric acid etc.) during operation of the processes, e.g.extraction, isolation or purification processes, the FR-900156 substancemay be transformed into and isolated in the form of the correspondingsalts thereof. Alternatively, thus prepared salts of the FR-900156substance can easily be converted to the free form, i.e. FR-900156substance per se in a conventional manner.

Further, the FR-900156 substance obtained in the free form may beconverted to the corresponding salts thereof with a base or an acid asmentioned above in a conventional manner.

Accordingly, it is to be understood that this invention includes withinthe scope thereof the FR-900156 substance as well as salts thereof asmentioned above.

The FR-900156 substance possesses the following physical and chemicalproperties (The following data are those of the product obtained byExample 94 (3):)

(1) Form and color: While powder.

(2) Nature of substance: Amphoteric.

(3) Color reaction:

Positive; each reaction with ninhydrin, potassium permanganate andsulfuric acid;

Negative; Dragendorff reaction and Ehrlich reaction.

(4) Solubility: Soluble; water; Sparingly soluble; methanol; Insoluble;ethanol, acetone, ethyl acetate, benzene, hexane, chloroform.

(5) MP; 143-148 (dec.).

(6) Specific rotation [α]_(D) ²⁵ =-27.1 (c=0.4 in water).

(7) Ultraviolet absorption spectrum: end absorption.

(8) Infrared absorption spectrum (KBr): 1050, 1130, 1235, 1340, 1400,1450, 1535, 1660, 1735, 2950, 2980, 3080, 3350 cm⁻¹.

(9) Elementary analysis: Qualitative analysis revealed that theFR-900156 substance comprises the following elements: Carbon, Hydrogen,Nitrogen and Oxygen.

(10) Thin layer chromatography:

    ______________________________________                                                        Developing                                                    Stationary phase                                                                              Solvent         Rf Value                                      ______________________________________                                        Eastman cellulose sheet*.sup.1                                                                BuOH,Acetic acid                                                                              0.35                                                          Water(4:1:2)                                                  Silicagel sheet Merck*.sup.2                                                                  60% i-propanol-water                                                                          0.65                                          ______________________________________                                         Note                                                                          *.sup.1 Trade name, made by Eastman Kodak Co.                                 *.sup.2 Trade name, made by Merck & Co.                                  

(11) Molecular weight: Mass spectrometory (Field desorption method):M=519 (base peak: M+1=520).

(12) Nuclear magnetic resonance absorption spectrum: (Solvent: D₂ O,Reference: TMSP).

(13) Amino acid analysis:

    ______________________________________                                                         Molar ratio                                                  ______________________________________                                        Glycine            1.00                                                       Glutamic acid      1.06                                                       Alanine            1.04                                                       α, ε-Diaminopimelic acid                                                           1.03                                                       ______________________________________                                         (Molar ratio is expressed as assuming Glycine = 1.00)                    

(14) Molecular formula: C₂₀ H₃₃ N₅ O₁₁.

Further, physical and chemical properties of more purified sample ofFR-900156 substance (i.e. a product obtained by Example for fermentation(4)) was measured, as a result of which MP of said sample is 147°-153°C. (dec.) and others properties thereof were the same as the above.

From analysis of the above physicochemical properties and the furtherinvestigation for elucidation of chemical structure, the chemicalstructure of the FR-900156 substance has been elucidated as follows:##STR23##

Preparation of Starting Compounds (II) and (III)

The starting Compounds (II) and (III) each include new compound and canbe prepared by methods as described below. Among the Compound (III), anew compound is represented by the following formula: ##STR24## wherein

R¹, R_(a) ², R_(b) ², R^(q) and R^(r) are each as defined above,

provided that when R¹ is hydrogen or t-butoxycarbonyl and R_(b) ² ishydrogen,

then R_(a) ² is mono- or di-carboxy lower alkyl having 1 and 3 to 6carbon atoms, α-carboxyethyl or ar(carboxy)lower alkyl whose aryl moietymay be substituted by hydroxy.

Detailed explanation of preparation of the starting Compounds (II) and(III) are made in the following.

Preparation of Compound (III), i.e. Compounds (III-1) to (III-15):##STR25## Preparation of Compound (II): ##STR26##

In the above formula,

R₂ ^(r) is an amino protective group,

R_(a) ^(2') is mono or di-protected carboxy-lower alkyl or ar(protectedcarboxy)lower alkyl whose aryl moiety may be substituted by hydroxy,

R₃ ^(q) is carboxy or protected carboxy excepting protected carbazoyl,

R₅ ^(q) is esterified carboxy, and R_(c) ¹, R_(b) ¹, R^(p), R₁ ^(r), R₁^(q), Y, m and n are each as defined above.

Particulars of the definitions for R₂ ^(r), and R_(a) ^(2') andpreferred example thereof are the same as those for R₁ ^(r) and R_(a) ²,respectively, and preferred example of esterified carboxy for R₅ ^(q) isthe same as those for R₃ ^(p), R_(c) ² and R₄ ^(q).

(1) Process 1^(s) : Compound (V-I)+Compound (IV)→Compound (III-1)

This process relates to a method for preparing Compound (III-1) byreacting Compound (V-1) or its salt with a Compound (IV) or its salt.

The starting Compound V-1) includes a known one described in e.g.Biochemistry, vol. 9, pages 823-831, 1971, and new one which can beprepared according to the method as described in said literature.

This reaction is carried out in substantially the same manner as that ofProcess 1 as explained hereinabove.

(2) Process 2^(s) : Compound (III-1)→Compound (III-2)

This process relates to a method for preparing Compound (III-2) bysubjecting Compound (III-1) to elimination reaction of both of aminoprotective groups for R₁ ^(r) and R₂ ^(r).

This process is applied to case that both amino protective groups for R₁^(r) and R₂ ^(r) are ones which can be removed by a method to beemployed in this process.

This reaction is carried out by conventional methods such as catalyticreduction method, liquid ammonia-alkalimetal method, acid method,zinc-acid method, base method, hydrazine method and the like. Amongthese methods, preferred one is selected according to kind of the aminoprotective groups for R₁ ^(r) and R₂ ^(r) of Compound (III-1).

This reaction is carried out in substantially the same manner as that ofProcess 2 explained hereinabove.

(3) Process 3^(s) : Compound (III-1)→Compound (III-3)

This process relates to a method for preparing Compound (III-3) or itssalt by removing selectively an amino protective group for R₂ ^(r) ofCompound (III-1) or its salt.

This process is applied in case that the amino protective group for R₂^(r) reveals a different chemical property from that for R₁ ^(r) againsteach kind of the removal methods as mentioned in the foregoing Process 2and can selectively be removable by a method to be employed.

This reaction is carried out by conventional methods as mentioned inexplanation for Process 2. Among these methods, preferred one isselected according to kinds of the amino protective group for R₂ ^(r).Each of such a method and the kinds of such amino protective group areto be referred to explanations in Process 2.

(4) Process 4^(s) : Compound (III-1)→Compound (III-4)

This process relates to a method for preparing Compound (III-4) or itssalt by removing selectively an amino protective group for R₁ ^(r) ofCompound (III-1) or its salt.

This process is applied to case that the amino protective group for R₁^(r) and the amino protective group of the protected carbazoyl for R₁^(q) reveals mutually the same chemical property and they reveal adifferent chemical property from that for R₂ ^(r) against each kind ofthe removal methods as mentioned in the foregoing Process 2 and they canselectively be removable by a method to be employed.

(5) Process 5^(s) : Compound (III-4)→Compound (III-5)

This process relates to a method for preparing Compound (III-5) or itssalt by removing an amino protective group for R₂ ^(r) or its salt.

This reaction is carried out in substantially the same manner as that ofProcess 3^(s) as explained hereinabove.

(6) Process 6^(s) : Compound (III-5)→Compound (III-6)

This process relates to a method for preparing Compound (III-6) byreacting Compound (III-15) with an amino protective agent.

The amino protecting agent includes organic carboxylic, sulfonic,sulfinic, phosphoric and carbonic acids, aldehyde and ketone compoundswhich are composed of the same protective group as explained for theamino protective group for R^(r), R₁ ^(r) and Y and their reactivederivatives.

The reactive derivatives include conventional ones such as halides,activated amides, activated esters, acid azide and the like.

When a free acid form of such an amino protecting agent is employed, thereaction is preferably carried out in the presence of a condensing agentconventionally used in the field of amino acid and peptide chemistry.

This reaction is carried out in water or an aqueous organic solvent inthe presence of a metal compound such as cupric chloride, cupric sulfatecupric carbonate, cupric acetate or the like.

(7) Process 7^(s) : Compound (III-5)→Compound (III-7)

This process relates to a method for preparing Compound (III-7) byreacting Compound (III-5) with an amino protecting agent.

The amino protecting agent to be used in this reaction is the same asone illustrated in Process 6^(s).

This reaction is carried out in a conventional solvent, preferablywithin the range of pH 6-9, more preferably at pH of around neutralityunder ice-cooling to at ambient temperature.

(8) Process 8^(s) : Compound (III-6)→Compound (III-10)

This process relates to a method for preparing Compound (III-10) byreacting Compound (III-6) with an amino protecting agent.

The amino protecting agent is the same as that of Process 6^(s) and thereaction condition is the same as those of Process 3 as explainedhereinabove.

(9) Process 9^(s) : Compound (VI)→Compound (VI-1)

This process relates to a method for preparing Compound (VI-1) byreacting Compound (VI) with an amino protecting agent.

In this reaction, it is necessary that such an amino protecting group tobe introduced into one of two amino groups of the Compound (VI) bedifferent from the amino protecting group for Y in case of obtainingstereospecific compound.

The amino protecting agent to be used is the same as the one illustratedin Process 6^(s).

This reaction is carried out in substantially the same manner as that ofProcess 6^(s).

(10) Process 10^(s) : Compound (VI)→Compound (VI-2)

This process relates to a method for preparing Compound (VI-2) byreacting Compound (VI) with an amino protecting agent.

In this reaction, such an amino protective group to be introduced intothe amino group may be the same as or different from that for Y.

Preferred examples of an amino protecting agent are the same as that ofProcess 6^(s).

This reaction is carried out in water or an aqueous organic solvent andpreferably within the range of pH 6-9, more preferably at pH of aroundneutrality under ice-cooling to at ambient temperature.

(11) Process 11^(s) : Compound (VI-1)→Compound (VI-3)

This process relates to a method for preparing Compound (VI-3) byreacting Compound (VI-1) with an amino protecting agnet.

In this reaction, it is necessary that such an amino protective group tobe introduced into the amino group is different from that for R₂ ^(r) ofCompound (VI-1) is case of obtaining stereospecific compound, but may bethe same as that for Y.

This reaction is preferably carried out at neutral-alkaline condition,and other reaction conditions are the same as that of Process 3 asexplained hereinabove.

(12) Process 12^(s) : Compound (VI-2)→Compound (VI-3)

This process relates to a method for preparing Compound (VI-3) byreacting Compound (VI-2) with an amino protecting agent.

In this reaction, it is necessary that such an amino protective group tobe introduced into the amino group (VI-3) is different from that for R₁^(r) and Y of Compound (VI-2) is case of obtaining stereospecificcompound.

Reaction conditions are the same as those of Process 11^(s).

(13) Process 13^(s) : Compound (VI-3)→Compound (III-8)

This process relates to a method for preparing Compound (III-8) byreacting Compound (VI-3) with a compound of the formula (IV): ##STR27##or its salt.

This reaction is carried out in substantially the same manner as that ofProcess 1 as explained hereinabove.

(14) Process 14^(s) : Compound (VI-3)→Compound (VI-4)

This process relates to a method for preparing Compound (VI-4) byremoving selectively an amino protective group for Y of Compound (VI-3).

This process is applied to case that the amino protective group for Yreveals a different chemical property from that for R₁ ^(r) and R₂ ^(r)against each kind of the removal methods as mentioned in the foregoingProcess 2 and can selectively be removed by a method to be employed.

This reaction is carried out in substantially the same manner as that ofProcess 2 as explained hereinabove.

(15) Process 15^(s) : Compound (VI-4)→Compound (III-11)

This process relates to a method for preparing Compound (III-11) byreacting Compound (VI-4) or its salt with a compound of the formula:##STR28## or its salt.

This reaction is carried out in substantially the same manner as that ofProcess 1^(s).

(16) Process 16^(s) : Compound (III-11)→Compound (III-15)

This process relates to a method for preparing Compound (III-5) byremoving both of amino protective groups for R₁ ^(r) and R₂ ^(r) ofCompound (III-11).

This reaction is carried out in substantially the same manner as that ofProcess 2^(s).

(17) Process 17^(s) : Compound (VI-5)→Compound (VI-6)

This process relates to a method for preparing Compound (VI-6) byreacting Compound (VI-5) with an esterifying agent.

An esterifying agent to be used in this reaction is the same as oneillustrated in the aforementioned Process 7 and the reaction is carriedout substantially in the same manner as that of Process 7.

(18) Process 18^(s) : Compound (VI-6)→Compound (VI-4)

This process relates to a method for preparing Compound (VI-4) byremoving selectively an amino protective group for Y of Compound (VI-6).

This reaction is carried out in substantially the same manner as that ofProcess 14^(s).

(19) Process 19^(s) : Compound (III-12)→Compound (III-13)

This process relates to a method for preparing Compound (III-13) byreacting Compound (III-12) with an esterifying agent.

An esterifying agent to be used in this process is the same as that ofProcess 17^(s).

This reaction is carried out in substantially the same manner as that ofProcess 17^(s).

(20) Process 20^(s) : Compound (III-13)→Compound (III-5)

This process relates to a method for preparing Compound (III-5) byremoving both of amino protective groups for R₂ ^(r) and R₁ ⁴ ofCompound (III-13).

This reaction is carried out in substantially the same manner as that ofProcess 16^(s).

(21) Process 21^(s) : Compound (V-2)→Compound (VI-7)

This process relates to a method for preparing Compound (VI-7) byreacting Compound (V-2) with a halogenating agnet.

A halogenating agent to be used in this reaction is to be such ahalogenating agent as being capable of halogenating a carboxy group toprovide the corresponding acid halide and may include a conventional onesuch as phorphorus trihalide (e.g. phosphorus trichloride, etc.),phosphorus pentahalide (e.g. phosphorus pentachloride, etc.), thionylhalide (e.g. thionyl chloride, etc.), phosgene and the like.

In this reaction, the followings are to be noted: That is, in case thata compound (V-2), wherein both of R^(r) and R^(r) are a carbonic acylgroup (e.g. alkoxycarbonyl, etc.), is employed as a starting compound,all of the halogenating agent as exemplified above can be used, and thereaction is conducted by reacting Compound (V-2) with the halogenatingagent under warming or heating. On the other hand, in case that acompound (V-2) wherein both of R^(r) and R^(r) are hydrogen, is employedas a starting compound, phosgene only is used as a halogenating agent,and the reaction is carried out by reacting Compound (V-2) with phosgeneunder cooling to warming.

(22) Process 22^(s) : Compound (VI-7)→Compound (VI-8)

This process relates to a method for preparing Compound (VI-8) byreacting Compound (VI-7) or its salt with a compound of the formula: H₂NNHY or its salt.

This reaction is preferably carried out in the presence of a weak acidsuch as acetic acid, oxalic acid or the like. This reaction is carriedout in a conventional solvent under ice-cooling to at ambienttemperature.

(23) Process 23^(s) : Compound (VI-8)→Compound (VI-9)

This process relates to a method for preparing Compound (VI-9) byreacting Compound (VI-8) with a conventional leucineaminopeptidase.

This reaction is preferably carried out in a conventional solvent, e.g.water, a hydrophilic solvent such as alcohol (e.g. methanol, ethanol,etc.), acetone or the like or a mixture thereof within the range of pH 7to 10, preferably pH 8 to 9, at 20° to 40° C., preferably 37° C.

(24) Process 24^(s) : Compound (VI-9)→Compound (VI-10)

This process relates to a method for preparing Compound (VI-10) with anamino protecting agent.

An amino protecting agent to be used are same as that of Process 6^(s).

This reaction is carried out in substantially the same manner as that ofProcess 3, as explained hereinabove.

(25) Process 25^(s) : Compound (VI-10)→Compound (VI-11)

This process relates to a method for preparing Compound (VI-11) byreacting Compound (VI-10) with a halogenating agent.

A halogenating agent is the same as that of Process 21^(s).

This reaction is carried out in substantially the same manner as that ofProcess 21^(s). In this respect, it is to be noted that, in case that acompound (VI-10), wherein R₂ ^(r) is a carbonic acyl (e.g.alkoxycarbonyl), is employed as a starting compound, all of thehalogenating agent as exemplified in the explanation for Process 21^(s)can be explained.

(26) Process 26^(s) : Compound (VI-11)→Compound (VI-12)

This process relates to a method for preparing compound (VI-12) bysubjecting compound (VI-11) to hydrolysis.

This reaction is carried out in substantially the same manner as that ofProcess 4-2 as explained hereinabove.

(27) Process 27^(s) : Compound (III-9)→Compound (III-9')

This process relates to a method for preparing compound (III-9') byreacting compound (III-9) with an esterifying agent.

An esterifying agent to be used is the same as that of Process 17^(s).

This reaction is carried out in substantially the same manner as that ofProcess 17^(s).

(28) Process 28^(s) : Compound (III-3)→Compound (III-14')

This process relates to a method for preparing compound (III-14') byreacting compound (III-3) with an amino protecting agent.

An amino protecting agent to be used is the same as that of Process6^(s).

This reaction is carried out in substantially the same manner as that ofProcess 8^(s).

(29) Process 29^(s) : Compound (III-14')→Compound (III-4)

This process relates to a method for preparing compound (III-4) byremoving selectively an amino protective group for R₁ ^(r).

This reaction is carried out in substantially the same manner as that ofProcess 4^(s).

(30) Process 30^(s) : Compound (III-4)→Compound (III-15)

This process relates to a method for preparing Compound (III-15) byreacting compound (III-4) with an amino protecting agent.

An amino protecting agent to be used is the same as that of Process6^(s).

This reaction is carried out in substantially the same manner as that ofProcess 8^(s).

(31) Process 31^(s) : Compound (III-15)→Compound (III-6)

This process relates to a method for preparing compound (III-6) byremoving selectively an amino protective group for R₂ ^(r).

This reaction is carried out in substantially the same manner as that ofProcess 3^(s).

(32) Process 32^(s) : Compound (VI-11)→Compound (III-16)

This process relates to a method for preparing compound (III-16) byreacting compound (VI-11) with a Compound of the formula: ##STR29## orits salt.

This reaction is carried out in substantially the same manner as that ofProcess 22^(s).

(33) Process 33^(s) : ##STR30##

This process relates to a method for preparing Compound (II) by reactingCompound (II-1) with an acylating agent.

The starting Compound (II-1) includes known one (e.g. Journal of Med.Chemistry, vol. 9, 971 (1966)) and new one, and said new compound can beprepared according to the method described in such literature.

The acylating agent to be used in this reaction is the same asillustrated in the aforementioned Process 3.

The acylation is carried out in substantially the same manner as that ofProcess 3 as explained hereinabove.

(34) Process 34^(s) : Compound (II-2)+Compound (II-3)→Compound (II)

This process relates to a method for preparing Compound (II) or its saltby reacting Compound (II-2) or its salt with Compound (II-3) or itssalt.

This reaction is carried out in substantially the same manner as that ofProcess 1 as explained hereinabove.

(35) Process 35^(s) : Compound (II-4)→Compound (II-5)→Compound (II)

This process relates to a method for preparing Compound (II) or its saltby reacting Compound (II-4) or its salt with Compound (II-5) or itssalt.

This reaction is carried out in substantially the same manner as that ofProcess 1 as explained hereinabove.

(36) Process 36^(s) : Compound (VI-3)→Compound (VI-2)

This process relates to a method for preparing Compound (VI-2) or itssalt by removing relectively an amino protective group for R₁ ^(r) ofCompound (VI-3).

This reaction is carried out in substantially the same manner as that ofProcess 3^(s) and accordingly the detailed explanation for Process 3^(s)as made hereinabove is to be referred thereto.

(37) Process 37^(s) : Compound (III-17)→Compound (III-18)

This process relates to a method for preparing Compound (III-18) byreacting Compound (III-17) with an oxidizing agent in the presence ofalcohol.

The oxidizing agent to be used in this process is the same as oneillustrated in Process 4-3.

The alcohol to be used in this process is a conventional one such asmethanol, ethanol, propanol, butanol, benzylalcohol or the like.

The reaction is carried out in substantially the same manner as that ofProcess 4-3.

(38) Process 38^(s) : Compound (III-19)→Compound (III-20)

This process relates to a method for preparing Compound (III-20) byreacting Compound (III-19) with an amino protecting agent.

An amino protecting agent to be used is the same as that of Process6^(s).

This reaction is carried out in substantially the same manner as that ofProcess 8^(s).

As to the object Compound (I) and starting Compounds (II) and (III)which are prepared according to the aforementioned Processes, it is tobe noted that each of said compounds includes one or more stereoisomerswhich in due to the asymmetric carbon atoms in their molecule and all ofsuch isomers are included within the scope this invention.

The new peptide (I) and its pharmaceutically acceptable salts of thisinvention have been found to possess enhancing activities of immuneresponse (i.e., enhancing activities of cellular immunity and humoralantibody production) and reticuloendotherial system, mitogenic activity,inducing activity of interferon, protective efficacy in experimentalinfection and anticancer activity.

Accordingly, the new peptide (I) and its pharmaceutically acceptablesalts are useful for the therapeutic treatment of infectious diseasescaused by pathogenic microorganism, especially gram-negative bacteriaand gram-positive bacteria and fugi, and of cancer in animals.

Further, Compounds (II) and (III) are useful as intermediate forpreparing Compound (I) having biologically active properties asmentioned above.

For the purpose of showing pharamaceutical utility of the new peptide(I), pharmacological test data thereof are illustrated in the following.

1. Blood Stream Clearance of Carbon

Reagents

1. Carbon suspension. Rotoring drawing ink (170 mg carbon/ml.) wasdiluted to 1/5 of the original concentration in saline containing 1%gelatin.

2. 0.1% aqueous sodium carbonate solution.

Procedure

Mice (DDY male 5-6 W) were injected via the tail vein with a dose of0.01 ml/g body weight of carbon.

Blood samples were taken by means of a pointed capillary pippetcalibrated to hold a 50 μl and previously washed in heparin. This wasplunged into the retrooribital venous sinus at the nasal angle of theeye. The samples were removed at 3 and 6 min. The blood was immediatelydischarged into 3.0 ml. of the sodium carbonate solution. Thishemolyszed the blood and allowed the quantitation of carbon. The sampleswere then read in a spectrophotometer at 660 nm, the log concentrationbeing obtained from a previously determined standard curve. Theclearance value K may be determined by plotting log carbon concentrationagainst time according to the following relationship; ##EQU1## in whichT₁ and T₂ represent the time in min when the sample were withdrawn andC₁ and C₂ represent the concentrations of carbon in the blood at thetime T₁ and T₂, respectively.

EXAMINATION OF EFFECT OF THE NEW PEPTIDE ON CARBON CLEARANCE

The aqueous solution of the test compound as given was administeredsubcutaneously to mice. Twenty four hours later, blood stream clearanceof carbon was measured. K value obtained with treated mice was comparedwith that of control mice. The test results are shown in Table 1 to 3.

                                      TABLE 1                                     __________________________________________________________________________    Test Compound (1)                                                              ##STR31##                                                                                                                             Reference                                                                     compound              R.sup.1        R.sub.b.sup.1                                                                           n                                                                               R.sub.a.sup.2                                                                            1*                                                                              2*                                                                              3*                                                                              4*                                                                              (mg/kg)Dose                                                                         ##STR32##                                                                          ##STR33##           __________________________________________________________________________     ##STR34##     CH.sub.3  1                                                                                ##STR35## L D L D  1 0.1                                                                              1.3 1.0                                                                             1.3 0.9             "              "         "                                                                                ##STR36## " " " "  1 1.0                                                                              1.6 1.1                                                                            1.3 0.9              "              "         "                                                                                ##STR37## " " " "  1 0.1                                                                              2.8 2.6                                                                            2.3 1.2              H              --        0 CH.sub.2 COOH                                                                            --                                                                              " " " 100   1.8  --                                                                  1    1.5  2.4                  CH.sub.3 CO    "         " "          " " " "  1    1.2  1.0                                                                 0.1  0.8  0.8                   ##STR38##     "         " "          " " " "  1. 0.1                                                                             1.8 1.9                                                                            1.8 1.9               ##STR39##     "         " "          " " " " 100  1                                                                              1.7 1.5                                                                            -- 1.8               H              "         "                                                                                ##STR40## " " " " 100  1                                                                              2.7 1.1                                                                            -- 1.8                ##STR41##     "         " "          " " " "  1 0.1                                                                              1.6 1.7                                                                            1.8 1.7              CH.sub.3 (CH.sub.2).sub.5 CO                                                                 "         " "          " " " "  1    1.9  1.8                                                                 0.1  2.5  1.7                  CH.sub.3 (CH.sub.2).sub.10 CO                                                                "         " "          " " " "  1    1.7  1.8                                                                  0.1 2.1  1.9                  CH.sub.3 (CH.sub.2).sub.16 CO                                                                CH.sub.3  1 CH.sub.2 COOH                                                                            " " " "  1    1.5  1.3                                                                 0.1  1.2  --                    ##STR42##     "         2 "          " " " "  1 0.1                                                                              1.3 1.5                                                                            1.3 0.9              CH.sub.3 CO    "         " "          " " " " 100   4.2  --                                                                  1    4.7  4.5                   ##STR43##     H         1 "          " " " " 100  1                                                                              2.0 0.9                                                                            -- 2.0               "              CH.sub.2 OH                                                                             " "          " " " " 100   2.3  --                                                                  1    1.1  2.0                                  ##STR44##                                                                              " "          " " " " 100  1                                                                              2.3 1.7                                                                            -- 2.0               "                                                                                             ##STR45##                                                                              " "          " " " " 100  1                                                                              2.9 1.6                                                                            -- 2.3                ##STR46##     CH.sub.3  " "          " " " "  1 0.1                                                                              1.4 1.8                                                                            1.8 1.9               ##STR47##     "         " "          " " " "  1 0.1                                                                              2.0 1.6                                                                            1.8 1.2               ##STR48##     "         " "          " " " " 100  1                                                                              2.2 1.5                                                                            -- 1.8                ##STR49##     "         " "          " " " "  1 0.1                                                                              1.2                                                                                1.8 1.7               ##STR50##     CO        " "          " " " "  1 0.1                                                                              2.0 1.3                                                                            1.8 1.9               ##STR51##     "         "                                                                                ##STR52## " " " "  1 0.1                                                                              1.8 1.5                                                                            1.8 1.7              "              "         "                                                                                ##STR53## " " " "  1 0.1                                                                              1.8 1.2                                                                            1.8 1.9              HOCH.sub.2 CO  CH.sub.3  1 CH.sub.2 COOH                                                                            " " " " 100   2.5  --                                                                  1    0.9  2.1                   ##STR54##     "         " "          " " " "  1 0.1                                                                              2.3 1.5                                                                            2.4 1.4               ##STR55##     "         " "          " " " " 100  1                                                                              2.0 1.8                                                                            -- 2.0                ##STR56##     "         " "          " " " " 100  1                                                                              1.6 1.9                                                                            -- 2.2                ##STR57##     "         " "          " " " " 100  1                                                                              2.1 1.9                                                                            -- 2.2                ##STR58##     "         " "          " " " " 100  1                                                                              1.6 2.2                                                                            -- 1.7                ##STR59##     "         " "          " " " " 100  1                                                                              1.9 0.7                                                                            -- 2.0               (CH.sub.3).sub.3 COO                                                                         "         " "          " " " " 100   2.9  --                                                                  1    2.3  1.8                   ##STR60##     "         " "          " " " " 100  1                                                                              2.1 2.0                                                                            -- 2.2               CH.sub.3 (CH.sub.2).sub.10 CO                                                                "         " "          " " " "  1    1.7  1.3                                                                 0.1  1.6  0.9                   ##STR61##     "         " "          " " " "  1 0.1                                                                              1.6 1.2                                                                            1.3 0.9               ##STR62##     "         " "          " " " "  1 0.1                                                                              3.0 1.7                                                                            3.6 1.6               ##STR63##     "         " "          " " " "  1 0.1                                                                              2.1 1.0                                                                            1.6 1.1              H              CH.sup.3  1 CH.sub.2 COOH                                                                            " " " " 100   2.1  --                                                                  1    0.8  2.1                   ##STR64##     "         " "          " " " " 100  1                                                                              1.0 0.7                                                                            -- 1.0               "              "         " "          " " " L  10   4.3  5.0                                                                 1    1.2  4.5                  "              "         " "          " L " D 100   2.8  --                                                                  1    1.4  3.6                   ##STR65##     "         " "          " D " " 100  1                                                                              2.6 2.4                                                                            -- 2.2                ##STR66##     "         " "          D " " "  1 0.1                                                                              2.2 1.9                                                                            1.8 1.2              CH.sub.3 CH.sub.2 CO                                                                         "         " "          L " " " 100   2.0  --                                                                  1    1.6  2.1                  CH.sub.3 CO    "         " "          " " " " 100   2.0  --                                                                  1    1.1  2.2                  CH.sub.3 (CH.sub.2).sub.5 CO                                                                 "         " "          " " " "  1    2.3  2.0                                                                 0.1  1.4  1.1                   ##STR67##     "         " "          " " " " 100  1                                                                              2.0 2.5                                                                            -- 1.7                ##STR68##     "         " "          " " " " 100  1                                                                              2.3 2.0                                                                            -- 2.6                ##STR69##     "         " "          " " " "  1 0.1                                                                              2.5 1.3                                                                            2.0 1.1               ##STR70##     "         " "          " " " " 100  1                                                                              1.5 1.3                                                                            -- 1.4                ##STR71##     "         " "          " " " " 100  1                                                                              1.5 1.3                                                                            -- 1.4               __________________________________________________________________________     Note:                                                                         ##STR72##                                                                

                  TABLE 2                                                         ______________________________________                                                        Dose                                                          Drug            (mg/mouse) K.sub.treated /K.sub.control                       ______________________________________                                        Krestin         10         1.1                                                                1          1.0                                                Levamisol       10         -- (death)                                                         1          0.5                                                Tuftsin         400        1.4                                                                125        1.0                                                FR-900156 substance                                                                           0.25       2.9                                                                0.06       1.5                                                Control (saline)           1.0                                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        (2) Test Compound 2                                                            ##STR73##                                                                                                      Reference                                                                     Compound                                    Test Compound                                                                             Dose                  FR-900156                                   *1  *2    *3    *4  *5  (mg/kg) K.sub.treated /K.sub.control                                                            substance                           ______________________________________                                        D   L     L     L   D   100     2.0       --                                                          1       0.9       1.9                                 D   D     L     L   D   100     1.7       --                                                                  1         1.6                                 D   L     D     L   L   100     2.7       --                                                          1       0.9       2.5                                 L   L     D     L   D   100     2.6       --                                                          1       2.4       2.2                                 D   D     D     L   D   1       2.2       1.8                                                         0.1     1.9       1.2                                 ______________________________________                                    

2. Enhancing Activities of Cellular Immunity and Humoral AntibodyProduction

Guinea pigs (groups of five) were given 0.1 ml of FIA (Freund'sIncomplete Adjuvant) emulsion containing 500 μg of ovalbumin in bothposterior footpads. Control groups received antigen in FIA only, whereasthe test groups received the antigen with the following test compoundsin FIA. The animals were skin-tested on day 14 and bled on day 16.

                                      TABLE 4                                     __________________________________________________________________________    (1) Test Compound (1)                                                          ##STR74##                                                                                                   Cellular                                                                              Humoral  immunity                                                     immunity*1                                                                            hemagglutination                                                                       hemolysin                                                 Dose                                                                             skin reaction                                                                         titer    titer                                                     (μg/                                                                          (mm diameter,                                                                         (M ± S.E.                                                                           (M ± S.E.                  R.sup.1       R.sub.b.sup.1                                                                       n R.sub.a.sup.2                                                                       site)                                                                            M ± S.E.)                                                                          log.sub.2)*2                                                                           log.sub.2)*2                  __________________________________________________________________________    H             CH.sub.3                                                                            1 CH.sub.2 COOH                                                                       1  1.8 ± 0.7*3                                                                        11.9 ± 0.5*3                                                                        5.6 ± 0.5                                              10 8.9 ± 0.9*3                                                                        12.3 ± 0.2*3                                                                        6.6 ± 0.3                                              100                                                                              10.8 ± 3.3*3                                                                       12.0 ± 0.3*3                                                                        6.7 ± 0.5                  CH.sub.3 CH.sub.2 CO                                                                        "     " "     1  2.0 ± 2.0                                                                          12.3 ± 0.2*3                                                                        7.3 ± 0.4*3                                            10 8.6 ± 1.0*3                                                                        11.4 ± 0.5*3                                                                        7.8 ± 0.3*3                                            100                                                                              3.1 ± 1.3*3                                                                        9.7 ± 0.4                                                                           7.2 ± 0.3*3                HOCH.sub.2 CO "     " "     1  10.4 ± 2.4*3                                                                       11.8 ± 0.5*3                                                                        7.4 ± 0.6*3                                            10 14.6 ± 1.1*3#2                                                                     12.4 ± 0.5*3                                                                        7.3 ± 0.5*3                                            100                                                                              6.6 ± 1.1*3                                                                        10.8 ± 0.2*3                                                                        6.0 ± 0.2                  Control                     0  0       9.4 ± 0.4                                                                           5.4 ± 0.5                   ##STR75##    "     " "     1 10                                                                             6.7 ± 0.8*3 10.2 ± 1.6*3                                                        8.2 ± 0.1*3 7.6 ± 0.5*3           ##STR76##    "     " "     1 10                                                                             8.7 ± 1.8*3 7.7 ± 2.4*3                                                         8.9 ± 0.2*3 8.7 ± 0.2*3          Control       "     " "     0  0       6.8 ± 0.3                            ##STR77##    "     " "     1 10 100                                                                         11.5 ± 2.1*3#1 11.1 ± 0.7*3#2 1.1                                       ± 1.1                                                                              10.8 ± 0.3*3 12.3 ± 0.5*3                                               9.1 ± 0.6                                                                           7.1 ± 0.4*3 8.5 ±                                                       0*3 4.3 ± 6*3              Control                     0  0       9.2 ± 0.3                                                                           5.9 ± 0.1                   ##STR78##    H     " "     1 10 100   10.0 ± 0.3*3 9.6 ± 0.3 10.0                                             ± 0.4 5.9 ± 0.3 6.9 ±                                                         0.5*3 7.0 ± 0.4*3                         ##STR79##                                                                          " "     1 10 100   10.6 ± 0.3*3 9.9 ± 1.0*3                                                10.5 ± 0.4*3                                                                        6.9 ± 0.3*3 7.0 ±                                                       0.4*3 6.3 ± 0.5*3          "             HOCH.sub.2                                                                          " "     1  10.1 ± 1.1*3#2                                                                     10.5 ± 0.2*3                                                                        7.8 ± 0.3*3                                            10 10.7 ± 1.3*3                                                                       9.8 ± 0.4                                                                           7.3 ± 0.3*3                                            100                                                                              5.4 ± 2.2                                                                          9.6 ± 0.4                                                                           7.1 ± 0.2*3                Control                     0  0.9 ± 0.9                                                                          8.5 ± 0.5                                                                           5.5 ± 0.3                   ##STR80##    CH.sub.3                                                                            " "     1 10                                                                             5.0 ± 2.2 7.1 ± 0.9*3                                                           8.5 ± 0.2*3 7.9 ± 0.4*3           ##STR81##    "     " "     1 10                                                                             5.4 ± 2.2*3 12.6 ± 0.9*3                                                        7.6 ± 0.4*3 8.4 ± 0.2*3          Control                     0  0       5.8 ± 0.3                           CH.sub.3 (CH.sub.2).sub.5 CO                                                                "     " "     1  10.0 ± 1.2*3                                                                       9.5 ± 0.4*3                                                     10 7.5 ± 2.2*3                                                                        9.2 ± 0.6*3                          ##STR82##    "     " "     1 10                                                                             5.5 ± 1.4*3 8.5 ± 1.0*3                                                         8.7 ± 0.5*3 7.4 ± 0.6*3          Control                     0  0       6.6 ± 0.6                            ##STR83##    "     " "     1 10                                                                             10.2 ± 1.1*3 6.3 ± 1.3*3                                                        8.3 ± 0.3*3 7.7 ± 0.3*3          CH.sub.3 CO   CH.sub.2                                                                            2 "     1  9.0 ± 1.0*3                                                                        7.7 ± 0.5                                         D             10 8.1 ± 2.5*3                                                                        8.4 ± 0.5*3                                       L                                                                ##STR84##    CH.sub.3                                                                            1 "     1 10                                                                             8.0 ± 1.1*3 11.9 ± 2.5*3                                                        8.8 ± 0.3*3 8.3 ± 0.3*3          Control                     0  0       7.1 ± 0.1                            ##STR85##    "     " "     0.1 1 10                                                                         11.2 ± 1.1*3 7.6 ± 2.2*3 6.4 ±                                       2.5     10.4 ± 0.3*3 10.2 ± 0.3*3                                               9.3 ± 0.2*3 -                        ##STR86##    "     " "     0.1 1 10                                                                         6.4 ± 2.7 10.2 ± 2.6*3 8.6 ±                                         1.8*3   9.9 ± 0.5*3 10.6 ± 0.3*3                                                10.6 ± 0.3*3                         ##STR87##    "     " "     0.1 1 10                                                                         10.5 ± 2.9*3 9.3 ± 0.7*3 4.2 ±                                       1.3     9.1 ± 0.4 9.1 ± 0.4 9.8 ±                                            0.2                                    Control                     0  0.9 ± 0.9                                                                          8.5 ± 0.4                           CH.sub.3 CO   --    0 "     1  6.4 ± 2.1*3                                                                        8.8 ±  0.3                                                      10 8.7 ± 1.0*3                                                                        9.1 ± 0.4                            ##STR88##    CH.sub.3                                                                            1                                                                                ##STR89##                                                                          1 10                                                                             13.7 ± 1.6*3 10.8 ± 0.9*3                                                       11.1 ± 0.5 10.7                     __________________________________________________________________________                                           ± 0.3                           

                                      TABLE 5                                     __________________________________________________________________________    (2) Test Compound (2)                                                          ##STR90##                                                                                   Cellular                                                                             Humoral  immunity                                                      immunity *1                                                                          Hemagglutination                                                                       hemolysin                                                     skin reaction                                                                        titer    titer                                          Test Compound                                                                           Dose (mm diameter,                                                                        (M ±  S.E.                                                                          (M ±  S.E.                                  *1                                                                              *2                                                                              *3                                                                              *4                                                                              *5                                                                              (μg/site)                                                                       M ± log.sub.2)*2                                                                           log.sub.2)*2                                   __________________________________________________________________________    D L D L D 0    0      9.1 ± 0.19                                                                          4.5 ± 0.45                                            0.1   8.2 ± 2.8*3                                                                       9.9 ± 0.10*3                                                                       6.4 ± 0.76                                            1    14.5 ± 2.1*3                                                                       11.5 ± 0.61*3                                                                       7.7 ± 0.64*3                                         10   11.0 ± 1.3*3                                                                       12.2 ± 0.56*3                                                                      7.1 ±  0.58                                           100   4.2 ± 1.7*3                                                                      10.0 ± 1.01                                                                         5.9 ± 0.89                                  D L L L D 0    0.9 ± 0.9                                                                         8.5 ± 0.4                                                                           --                                                       1    2.4 ± 1.5                                                                         10.4 ± 0.5*3                                                                        --                                                       10    7.9 ± 2.1*3                                                                      11.0 ± 0*3                                                                          --                                             D L D     0    0      9.2 ± 0.3                                                                           5.9 ± 0.1                                         DL  1     4.3 ± 1.8*3                                                                      10.2 ± 1.1*3                                                                        6.5 ± 0.4                                             10    9.0 ± 3.2*3                                                                      10.4 ± 0.3*3                                                                         7.5 ± 1.2*3                                          100   6.4 ± 1.7*3                                                                      10.9 ± 0.2*3                                                                         6.7 ± 1.2*3                                L L D L D 0    0      6.8 ± 0.3                                                                           --                                                       1     6.7 ± 0.8*3                                                                       8.2 ± 0.1*3                                                                        --                                                       10   10.2 ± 1.6*3                                                                       7.6 ± 0.5*3                                                                        --                                             __________________________________________________________________________     Note:                                                                         *1: The skin test was performed on the back by intradermal injection of 5     μg of antigen dissolved in 0.1 ml of saline. Skin reaction of the test     site was measured at 48 hours.                                                *2: Antibody estimation was carried out as follows:                           Ovalbumin-coated sheep red blood cells were prepared by chromium chloride     Antibody titer was expressed as the reciprocal of the highest dilution of     serum evoking threshold hemogglutination and hemolysin.                       The results were converted to log.sub.2 unit.                                 *3: Significance was calculated by Student's ttest; P<0.05                    #1: 2/5 of animal, Central necrosis                                           #2: 1/5 of animal, Central necrosis                                      

3. Mitogenic Activities for Mouse Spleen Cells (Materials and Methods)

(1) Animal:

Mice used for this experiment were male BALB/C Strain, aged 13 weeks(Test 1) or female BALB/C strain, aged 9 weeks (Test 2).

(2) Tissue Culture Medium:

The tissue culture medium employed was a complete medium designatedRoswell Park Memorial Institute (RRMI)-1640. All media employedcontained 100 units/ml of penicillin G and 100 μg/ml of streptomycinsulfate and 10% fetal calf serum.

(3) Cell Preparation:

Spleens were removed under sterile conditions, and washed with Hankssolution and then teased in the tissue culture medium. The cells weresuspended in the tissue culture medium to contain 8×10⁶ cells/ml.

(4) Culture Conditions:

Into each hole of Microtest II tissue culture plate (8×12 hole) (maker:Falcon Plastics Co.) were poured 0.1 ml of the above cells suspensionand 0.1 ml of the prescribed concentrate of the test compound asdescribed below and then the cultures were incubated intriplicate at 37°C. in a humidified atmosphere (95% air, 5% CO₂) for 48 hours.

The control culture contained 0.1 ml of the culture medium instead ofthe medium containing the test compound.

(5) (3H) Thymidine uptake:

In all tests, 20 μl of 10 micro-curine (μCi)/ml of tritiated thymidine(3H-thymidine) was added to each hole for the final 24 hours of culture.After the culture was completed, the resultant cells were filtered witha filter paper, Whatman GF83 and washed successively with saline andwith 5% trichloroacetic acid. The filter paper was dried and placed in ascintillator (toluene 1 l, containing 0.1 g ofp-bis(5-phenyloxazoyl)benzene and 4 g of 2,5-diphenyloxazoyl), and3H-thymidine incorporated into DNA was measured.

(6) Stimulation Index ##EQU2##

                                      TABLE 6                                     __________________________________________________________________________    (7) Test Compound (1)                                                         (Results)                                                                     (1) Test 1                                                                                                 3Hthymidine                                                           Concentration                                                                         uptake net                                                                            Stimulation                              R.sup.1     R.sup.2  (μg/ml)                                                                            cpm: av ± S.E.                                                                     Index                                    __________________________________________________________________________    CH.sub.3 (CH.sub.2).sub.5 CO                                                              CH.sub.3 100     2,728 ± 73                                                                         5.2                                                            10     2,660 ± 45                                                                         5.0                                                            1      2,899 ± 126                                                                        5.5                                       ##STR91##                                                                                 ##STR92##                                                                             100  10 2,174 ± 142 1,547 ± 79 1,103 ±                                               4.1 2.9 2.1                               ##STR93##  CH.sub.3 100  10 2,234 ± 170 2,046 ± 138 1,378 ±                                              4.2 3.9 2.6                               ##STR94##  CH.sub.3 100  10 2,312 ± 158 2,388 ± 97 1,775 ±                                               4.4 4.5 3.4                              Control     --       --        528 ± 28                                                                         1.0                                       ##STR95##  CH.sub.3 100  10 2,306 ± 54 1,788 ± 314 1,928 ±                                               5.5 4.3 4.6                              HOCH.sub.2 CO                                                                             CH.sub.3 100     2,605 ± 117                                                                        6.2                                                            10     1,725 ± 86                                                                         4.1                                                            1      1,120 ± 55                                                                         2.7                                      CH.sub.3 CH.sub.2 CO                                                                      CH.sub.3 100     2.023 ± 142                                                                        4.9                                                            10     1,474 ± 44                                                                         3.5                                                            1        732 ± 28                                                                         1.8                                       ##STR96##  H        100  10 1,541 ± 73 1,119 ± 63   937 ±                                                3.7 2.7 2.2                               ##STR97##                                                                                 ##STR98##                                                                             100  10 1,675 ± 22 1,679 ± 98 1,019                                                     4.0 4.0 2.4                               ##STR99##  HOCH.sub.2                                                                             100  10 1,541 ± 41 1,468 ± 12   927 ±                                                3.7 3.5 2.2                               ##STR100## CH.sub.3 100  10 1,635 ± 192 1,573 ± 69 1,001 ±                                               3.9 2.8 2.4                              Control     --       --        417 ± 13                                                                         1.0                                       ##STR101## CH.sub.3 100  10 1,979 ± 174 2,343 ± 22 1,469 ±                                               5.0 5.9 3.7                               ##STR102## CH.sub.3 100  10 1,537 ± 113 1,788 ± 30 1,144 ±                                               3.9 4.5 2.9                               ##STR103## CH.sub.3 100  10 1,627 ± 38 1,837 ± 138 1,338 ±                                               4.1 4.6 3.4                               ##STR104## CH.sub.3 100  10 1,528 ± 122 1,225 ± 42   866 ±                                               3.8 3.1 2.2                               ##STR105## CH.sub.3 100  10 1,588 ± 167 1,469 ± 87 1,065 ±                                               4.0 3.7 2.7                              Control              --        399 ± 51                                                                         1.0                                      __________________________________________________________________________

                  TABLE 7                                                         ______________________________________                                        (2) Test 2                                                                                           Con-                                                                          cen-   3Hthymi-                                                               tra-   dine                                                                   tion   uptake net                                                                             Stimu-                                                        (μg/                                                                              cpm:     lation                                 R.sup.1         R.sup.2                                                                              ml)    av ± S.E                                                                            Index                                  ______________________________________                                        CH.sub.3 CO     CH.sub.3                                                                             100    1,920 ± 135                                                                         4.2                                                            10    1,067 ± 63                                                                          2.4                                                            1       785 ± 33                                                                          1.7                                     ##STR106##     CH.sub.3                                                                             100  10  1                                                                           2,772 ± 215 2,326 ± 140 1,713 ±                                      102      6.1 5.1 3.8                             ##STR107##     CH.sub.3                                                                             100  10  1                                                                           2,979 ± 199 2.177 ± 66 1,477 ±                                                6.6 4.8 3.3                             ##STR108##     CH.sub.3                                                                             100  10  1                                                                           2,186 ± 184 1,370 ± 58   756 ±                                                4.8 3.0 1.7                            Control                --       452 ± 30                                                                          1.0                                     ##STR109##     CH.sub.3                                                                             100  10  1                                                                           4,227 ± 217 3,664 ± 122 2,809 ±                                               4.2 3.7 2.8                            CH.sub.3 (CH.sub.2).sub.10 CO                                                                 CH.sub.3                                                                             100    4,027 ± 87                                                                          4.0                                                            10    3,963 ± 198                                                                         4.0                                                            1     4,210 ± 255                                                                         4.2                                     ##STR110##     CH.sub.3                                                                             100  10  1                                                                           3,967 ± 348 2,933 ± 126 2,134 ±                                               4.0 2.9 2.1                            Control                --     1,000 ± 94                                                                          1.0                                    ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        (8) Test Compound (2)                                                          ##STR111##                                                                                      Concen-  3Hthymidine                                                                             Stimu-                                  Test Compound      tration  uptake net                                                                              lation                                  *1  *2    *3    *4  *5  Test (μg/ml)                                                                           cpm: av ± S.E.                                                                       Index                           ______________________________________                                        D   L     D     L   D   1    100    2,028 ± 89                                                                           4.9                                                          10     1,486 ± 120                                                                          3.6                                                          1        835 ± 64                                                                           2.0                                                          0        417 ± 13                                                                           1.0                                                     2    100    3,666 ± 42                                                                           4.1                                                          10     3,223 ± 402                                                                          3.6                                                          1      2,741 ± 319                                                                          3.0                                                          0        901 ± 105                                                                          1.0                             D   L     D     .BHorizBrace.                                                                       1    100    1,284 ± 131                                                                          3.1                                               DL         10     1,414 ± 38                                                                           3.4                                                            1        996 ± 35                                                                           2.4                                                          0        417 ± 13                                                                           1.0                             L   L     D     L   D   2    100    2,772 ± 215                                                                          6.1                                                          10     2.326 ± 140                                                                          5.1                                                          1      2,713 ± 102                                                                          3.8                                                          0        452 ± 30                                                                           1.0                             D   L     D     L   L   1    100    1.271 ± 29                                                                           3.2                                                          10     1,003 ± 50                                                                           2.5                                                          1        742 ± 61                                                                           1.9                                                          0        399 ± 51                                                                           1.0                             ______________________________________                                    

4. Protective Efficacy in Experimental Infection in Mice

In determining the protective efficacy against experimental infectionsin mice, the test compound was dissoloed in and diluted with sterilesaline to provide prescribed concentrations of drug.

Male ICR-strain mice, aged 4 weeks were used in groups of ten mice. E.coli 22 was cultivated overnight at 37° C. on trypticase soy agar andthen were suspended in a sterile saline to obtain microbial cellconcentration of 9.0×10⁷ CFU/ml. Mice were inoculated intraperitoneallywith 0.2 ml of the suspension. Each of the test drugs were givenintraperitoneally in various doses to a group of ten mice 24 hoursbefore challenge.

Survival percent were found from the number of the surviving animalsafter four days of injection. Results are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Test Compound (1):                                                             ##STR112##                    (1)                                                                   Survival (%)                                                                        Dose    Dose                                                                  10 mg/  1 mg/                                    R.sup.1         R.sup.2      kg      kg                                       ______________________________________                                        CH.sub.3 (CH.sub.2).sub.5 CO                                                                  CH.sub.3     60      70                                       (CH.sub.3).sub.3 CCO                                                                          CH.sub.3     70      70                                       CH.sub.3 (CH.sub.2).sub.10 CO                                                                 CH.sub.3     50      60                                        ##STR113##     CH.sub.3     100     88.9                                      ##STR114##     CH.sub.3     88.9    100                                       ##STR115##     CH.sub.3     100     88.9                                      ##STR116##     CH.sub.3     87.5    75.0                                      ##STR117##     CH.sub.3     30      50 -                                      ##STR118##     CH.sub.3     20      60                                        ##STR119##     CH.sub.3     50      30                                        ##STR120##     CH.sub.3     70      70                                        ##STR121##     CH.sub.3     50      50                                        ##STR122##     CH.sub.3     50      50                                        ##STR123##     CH.sub.3     80      60                                        ##STR124##                                                                                    ##STR125##  60      60                                        ##STR126##     CH.sub.3     40      70                                       HOCH.sub.2 CO   CH.sub.3     75      100                                      CH.sub.3 CH.sub.2 CO                                                                          CH.sub.3     100     87.5                                      ##STR127##                                                                                    ##STR128##  87.5    100                                       ##STR129##     HOCH.sub.2   87.5    87.5                                      ##STR130##     H            100     100                                       ##STR131##     CH.sub.3     87.5    62.5                                      ##STR132##     CH.sub.3     100     87.5                                      ##STR133##     CH.sub.3     100     87.5                                     CH.sub.3 CO     CH.sub.3     62.5    25.0                                     ______________________________________                                    

                                      TABLE 10                                    __________________________________________________________________________    (2) Test Compound (2)                                                          ##STR134##                                                                                                       Survival (%)                                                                  Dose Dose                                 R.sup.1      R.sub.b.sup.1                                                                    n R.sub.a.sup.2                                                                           *1                                                                              *2                                                                              *3                                                                              *4                                                                              10 mg/kg                                                                           1 mg/kg                              __________________________________________________________________________     ##STR135##  CH.sub.3                                                                         1 CH.sub.2 COOH                                                                           L D L D 75.0 50.0                                  ##STR136##  CH.sub.3                                                                         1 CH.sub.2 COOH                                                                           L D L D 87.5 75.0                                  ##STR137##  "  " "         " " " " 75.0 87.5                                  ##STR138##  "  " "         " " " " 75.0 87.5                                  ##STR139##  "  " "         " " " " 87.5 62.5                                  ##STR140##  "  "                                                                                ##STR141##                                                                             " " " " 87.5 87.5                                  ##STR142##  "                                                                                   ##STR143##                                                                             " " " " 70.0 70.0                                 "            "  "                                                                                ##STR144##                                                                             " " " " 90.0 80.0                                 "            "  "                                                                                ##STR145##                                                                             " " " " 60   60                                   H            -- 0 CH.sub.2 COOH                                                                           --                                                                              " " " 66.6 22.2                                 CH.sub.3 CO  "  " "         " " " " 100  88.9                                  ##STR146##  "  " "         " " " " 87.5 75.0                                  ##STR147##  -- 0 CH.sub.2 COOH                                                                           --                                                                              D L D 87.5 50.0                                 H            "  "                                                                                ##STR148##                                                                             " " " " 75.0 87.5                                  ##STR149##  "  " "         " " " " 62.5 50.0                                 CH.sub.3 (CH.sub.2).sub.5 CO                                                               "  " "         " " " " 62.5 62.5                                 CH.sub.3 (CH.sub.2).sub.10 CO                                                              "  " "         " " " " 87.5 100                                  H            "  " CH.sub.2 COOH                                                                           " " " " 87.5 100                                   ##STR150##                                                                                CH.sub.3                                                                         1 "         L " " " 87.5 50.0                                  ##STR151##  "  " "         " " " L 100  88.9                                 "            "  " "         " L " D --   50.0                                  ##STR152##  "  " "         D " " " 100  87.5                                  ##STR153##  CH.sub.3                                                                         1 CH.sub.2 COOH                                                                           D D L D 75.0 75.0                                 CH.sub.3 (CH.sub.2).sub.5 CO                                                               "  "                                                                                ##STR154##                                                                             L " " " 100  44.4                                 __________________________________________________________________________

                  TABLE 10-a                                                      ______________________________________                                        (2) Test Compound (2)                                                          ##STR155##                                                                                            Survival                                                                                        Dose Dose                                                                     10   1                                                                        mg/  mg/                           R.sup.1 R.sub.b.sup.1                                                                        n     R.sup.2  *1  *2  *3 *4                                                                              kg   kg                            ______________________________________                                        CH.sub.3 (CH.sub.2).sub.5                                                             CO                                                                            CH.sub.3                                                                             1     COOH     L   D   L D  66.6 88.9                          "       --     0     "        --  "   " "  88.9 100                           H       "      "     "        "   "   " "  88.9 88.9                           ##STR156##                                                                           CH.sub.3                                                                             1     "        L   "   " "  100  62.5                          "       "      "                                                                                    ##STR157##                                                                            "   "   " "  87.5 75.0                          "       "      "     H        "   "   -- " 40.0 60.0                          ______________________________________                                    

(2) In determining the protective efficacy against experimentalinfections in mice, the FR-900156 substance was dissolved and diluted insterile water to provide three-fold concentrations of drug for testing.

Male DDY-strain mice, aged 6 weeks and averaging 24-26 g in weight, wereused in groups of four mice each.

Overnight cultures of Escherichia coli No. 22 in Difco Nutrient Brothwas diluted to 1/100 in fresh medium and incubated at 30° C. withshaking. When the cell density of 1×10⁸ /ml was obtained, 0.2 ml of theculture was injected intraperitoneally. All the animals receiving thechallenge and not treated with the drug died within 48 hours of theinfection.

One-fifth ml of the FR-900156 substance solution was injectedsubcutaneously, 1,4,5, and 6 days before the infection.

Two days after infection, the test was considered complete and survivalrecords of that day were made. The test results are shown in Table 11.

                  TABLE 11                                                        ______________________________________                                        Dose (mg/mouse/day)                                                                            Survival/infected                                            ______________________________________                                        0.01             10/10                                                        0.003            10/10                                                        0.001            10/10                                                        0.0003            8/10                                                        0.0001            4/10                                                        Control           0/10                                                        ______________________________________                                    

5-1. Anticancer Activity

Female rats of Donru Strain, aged 6 weeks, were used in groups of threerats each.

Suspension of ascites hepatoma AH 66 in 0.5 ml of Hank's solution wastransplanted intraperitoneally (5×10⁴ cells/rat). About 13 days later,all the control animals dies of ascites. Prolongation of survival timein comparison with the controls is the criterion of effectiveness.

Therapy was given 5, 6, 7, 8, 9, days before tumor transplantation. TheFR-900156 substance was dissolved and diluted in sterile saline water toprovide three-fold dilutions for testing and the said sterile salinesolution of the FR-900156 substance was given intraperitoneally to theanimals. The test results are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                        Dose (mg/rat/day)                                                                             Life span (days)                                              ______________________________________                                        1.0             14, >30, >30                                                  0.3             13, >30, >30                                                  0.1             13, >30, >30                                                   0.03           13,  13,  13                                                  Control (saline)                                                                              13,  13,  13                                                  ______________________________________                                    

5-2. Antitumor Activity

Methylcholanthlene-induced fibrosarocoma (Meth-A) was used.

A mixture of the tumor cells (1×10⁵) and the test compound was suspendedin a 0.5% methylcellulose saline solution. The suspension was inoculatedintradermally in male BALB/c mice.

Four weeks after inoculation, tumor size was measured. Results are shownin the following table.

    ______________________________________                                         ##STR158##                                                                                                  Suppres-                                                               Dose   sion of                                        Test compound           μg/ Meth-A                                         R.sup.1      R.sup.p  R.sub.a.sup.2                                                                        R.sup.q                                                                            site growth                                 ______________________________________                                        CH.sub.3 (CH.sub.2).sub.16 CO                                                              H        H      H     10  8/10                                   Control                           --   1/20                                   CH.sub.3 (CH.sub.2).sub.16 CO                                                              H        CH.sub.3                                                                             CH.sub.3                                                                           100  10/10                                  Control                           --   1/20                                   [CH.sub.3 (CH.sub.2).sub.21 ].sub.2 CHCO Control                                            ##STR159##                                                                            CH.sub.3                                                                             CH.sub.3                                                                           100 --                                                                             7/10 1/20                              ______________________________________                                         Note:-                                                                        Values gives Number of tumorfree mice/Number of mice tested, provided tha     when the perpendicular diameter of the tumor was less than 5 mm, the          animal was defined as free of tumor.                                     

6. Inducing Activity of Interferon ##STR160##

Male ICR strain mice aged 4 to 6 weeks were used in these experiments.Spleens of these mice were removed and the capacity of spleen cells toproduce interferon in vitro by the treatment of test compound wastested.

Single spleen cell subpensions were prepared in medium RPMI-1640supplement with streptomycin (100γ/ml), penicillin G (100γ/ml), 1%glutamine and 2-mercaptoethanol (5×10⁻⁵ M).

These were cultured at 37° C. at a concentration of 4×10⁷ cells/ml withor without test compound dissolved with culture medium (dose: 100, 10, 1μg/ml). After 24 hours, supernatants of these cultures were collectedand those interferon activities were titrated by cytopathic effect (CPE)inhibition test in L cell-VSV system. Antiviral titer was expressed inIU/ml base on standard interferon.

The results are as the following Table 13.

                  TABLE 13                                                        ______________________________________                                        Test Compound    Dose                                                         *1    *2    *3      *4  *5   (μg/ml)                                                                           IP Titer (IU/ml)                          ______________________________________                                        D     L     D       L   D    100    14                                                                     10     16                                                                      1     10                                                                      0     <6.5                                      D     L     D       .BHorizBrace.                                                                        100    62                                                              DL     10     40                                                                      1     39                                                                      0     7.5                                         D     D     D       L   D    100    29                                                                     10     23                                                                      1     11                                                                      0     <11                                       D     D     L       L   D    100    38                                                                     10     11                                                                      1     <11                                                                     0     <11                                       ______________________________________                                    

7. Acute Toxicity in Mice of FR-900156: 1 g/kg (i.v.): no toxicity

8. Cell Toxicity (mouse L cell) of FR-900156: 500 μg/ml: no toxic effect

The pharmaceutical composition of this invention can be used in the formof a pharmaceutical preparation, for example, in solid, semisolid orliquid form, which contains an active substance of this invention inadmixture with an organic or inorganic carrier or excipient suitable forexternal, enteral or parenteral applications. The active ingredient maybe compounded, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, suppositories,solutions, emulsions, suspensions, and any other form suitable for use.The carriers which can be used are water, glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, collidal silica, potato starch, urea and other carrierssuitable for use in manufacturing preparations, in solid, semisolid, orliquid form, and in addition auxiliary, stabilizing, thickening andcoloring agents and perfumes may be used. The pharmaceuticalcompositions can also contain preservative or bacteriostatic agents tokeep the active ingredient in the desired preparations stable inactivity. The active object compound is included in the pharmaceuticalcomposition in an amount sufficient to produce the desired therapeuticeffect upon the process or condition of diseases.

For applying this composition to humans, it is preferably to apply it byintravenous, intramuscular or oral administration. While the dosage ortherapeutically effective amount of the object compound of thisinvention varies from and also depends upon the age and condition ofeach individual patient to be treated, a daily dose of about 2-100 mg ofthe active ingredient/kg of a human being or an animal is generallygiven for treating diseases, and an average single dose of about 50 mg,100 mg, 250 mg, and 500 mg is generally administered.

The following examples are given for purpose of illustrating thisinvention.

In the following examples, starting compounds and object compounds areexpressed by using the following abbreviations:

Lac: Lactyl

Ala: Alanyl

Glu: Glutamyl

Gly: Glycyl

DAP: α,ε-Diaminopimelyl

Z: benzyloxycarbonyl

Boc: t-butoxycarbonyl

Bzl: benzyl

Me: methyl

Et: ethyl

Su: N-hydroxysuccinimide

Bzh: benzhydryl

Ac: acetyl

Val: Valyl

Ser: Seryl

Phe: Phenylalanyl

Asp: Aspartyl

Lys: Lysyl

Leu: Leucyl

Sar: Sarcosyl

γ-Abu: γ-Aminobutyryl

Aad: 2-Aminoadipyl

Tyr: Tyrosyl

Tfa: Trifluoroacetyl

Preparation 1 ##STR161##

Meso-DAP(D)-NHNH.Boc (1)(5.4 g) and cupric chloride (dihydrate)(1.52 g)were dissolved in a mixture of water (30 ml) and 0.5N aqueous sodiumhydroxide (60 ml). The solution was stirred for thirty minutes atambient temperature. The solution was cooled to 0° C. andbenzyloxycarbonyl chloride (4.6 g) was added dropwise thereto in thecourse of twenty minutes, maintaining the pH over 11. The stirring wascontinued for an additional one hour at the same temperature. After thereaction was completed, the solution was adjusted to pH 7 with 5%aqueous hydrochloric acid and they hydrogen sulfide gas was passedthrough the solution for about fifteen minutes. To the reaction mixturewas added ethyl acetate (50 ml) and the mixture was adjusted to pH 4with 5% aqueous hydrochloric acid. The ethyl acetate layer and theaqueous layer were divided out, and the ethyl acetate layer wasextracted four times with water (30 ml). All of the aqueous layers werecombined and concentrated to 40 ml under reduced pressure to give acrystals. The crystals were collected by filtration, washed with waterto give Z-(L)meso-DAP(D)-NHNH.Boc (Z)(3.5 g). The mother liquor and thewashings were collected and extracted twice with n-butanol (40 ml). Theextracts were combined and concentrated under reduced pressure to give aresidue. The residue was triturated with ether and collected byfiltration to give additional Z-(L)-meso-DAP(D)-NHNH.Boc (2)(1.2 g) as acrude solid, which was recrystallized from water. mp 194-196 (dec.).

N.M.R. (CD₃ OD), δ(ppm): 1.67 (9H, s), 1.6-2.1 (6H, m), 3.6-4.2 (2H, m),5.12 (2H, s), 7.43 (5H, s).

[α]_(D) =-20.8°(C:0.5 methanol).

Preparation 2 ##STR162##

3N Aqueous sodium hydroxide (6.0 ml) was added to a solution ofmeso-DAP-(D)-GlyOH (1)(1.85 g) and cupric chloride (dihydrate)(1.28 g)in water (190 ml) at 5° C. To the solution was added benzyloxycarbonylchloride (5.4 ml), and the resulting solution was stirred at 5° C.,maintaining the pH 11-12 with 3N aqueous sodium hydroxide. The stirringwas continued for 4.5 hours and an additional benzyloxycarbonyl chloride(3.2 ml) was added to the reaction mixture. The mixture was stirred at5° C. for six hours, maintaining the pH 11-12 with 3N aqueous sodiumhydroxide.

The reaction mixture was acidified to pH 2 with 3N aqueous hydrochloricacid and washed twice with ether. The separated aqueous layer wasadjusted to pH 4.5 with 3N aqueous sodium hydroxide and buffled withhydrogen sulfide and then filtered. The filtrate was concentrated toabout 100 ml and the concentrate was allowed to stand in a refrigeratorto give crystals. The crystals were collected by filtration and washedsuccessively with water, methanol and ether to giveZ-(L)-meso-DAP-(D)-GlyOH (2)(230 mg). mp 161.5-162.0 (dec.)

N.M.R. (CD₃ OD--D₂ O) δ(ppm): 1.2-2.2 (6H, m), 3.7-4.3 (4H, m), 5.10(2H, s), 7.39 (5H, s), [α]_(D) =-25.3° (C:0.5 methanol).

The filtrate was concentrated and chromatographed on a macroporousnon-ionic adsorption resin, HP20 (trade mark, maker: Mitsubishi ChemicalIndustry Co., Ltd.) (125 ml). After washing with water, elution wascarried out with 50% aqueous methanol and then 80% aqueous methanol. Theeluates were combined and evaporated to dryness and the crude crystalsthus obtained was dissolved in hot methanol (80 ml) and filtered. To thefiltrate was added water (15 ml) and the solution was concentrated togive crude crystals. The crude crystals were washed successively withisopropanol and ether to givel Z-(L)-meso-DAP-(D)-GlyOH (2)(2.22 g). Thesame object compound (2)(165 mg) was additionally obtained from themother liquor.

Preparation 3 ##STR163##

meso DAP(OH)GlyOEt (1) (4.10 g) was dissolved in water (30 ml) andcupric chloride (dihydrates)(2.08 g) was added to the solution. Themixture was stirred at 5°-7° C. for five hours, maintaining pH 11 with2% aqueous sodium hydroxide.

To the reaction mixture was added benzyloxycarbonyl chloride (10.0 g)and the resulting mixture was stirred at 5°-7° C. for five hours,maintaining pH 11. An additional benzyloxycarbonyl chloride (4.0 g) wasadded to the reaction mixture. The resulting mixture was stirred forfour hours and adjusted to pH 1 with dil hydrochloric acid and washedwith ethyl acetate. The aqueous layer was adjusted to pH 7 and hydrogensulfide gas was passed through the solution. The precipitates werefiltered off and the filtrate was concentrated. The concentrate wasadjusted to pH 3.2-3.4 and passed through a column packed with amacroporous non-ionic adsorption resin, HP20 (180 ml). Elution wascarried out with 50% aqueous methanol and the eluate was evaporated todryness to give Z-meso DAP(OH)GlyOH (2)(4.3 g) as a solid.

N.M.R. (D₂ O+NaHCO₃) δ(ppm): 1.38-1.88 (6H, m), 3.75 (2H, s), 3.79 (1H,t, J=4 Hz), 3.96 (1H, t, J=4 Hz), 5.11 (2H, s), 7.44 (5H, s).

Preparation 4 ##STR164##

Cupric carbonate (1.173 g) was added to a solution of DAP(OH)GlyOH(1)(1.7 g) in water (50 ml) and the mixture was refluxed for two hours.The reaction mixture was filtered and the filtrate was adjusted to pH11.5 with 2N aqueous sodium hydroxide. Benzyloxycarbonyl chloride (3.23ml) was added to the reaction mixture at 0° C. and the resulting mixturewas stirred at 0° C. for six hours, maintaining pH 11.5. The reactionmixture was neutralized with dil. aqueous hydrochloric acid anddechelated by hydrogen sulfide gas. The resulting mixture was filteredand the filtrate was chromatographed on a macroporus non-ionicadsorption resin, HP20 (100 ml). Elution was carried out with 80%aqueous methanol and the eluate was evaporated to dryness to giveZ-DAP(OH)GlyOH (2)(1.4 g) as a solid.

N.M.R. (CD₃ OD) δ(ppm): 1.38-1.88 (6H, m), 3.75 (2H, s), 3.79 (1H, t,J=4 Hz), 3.96 (1H, t, J=4 Hz), 5.11 (2H, s), 7.44 (5H, s).

Preparation 5 ##STR165##

(1) Preparation 5-1

Z-(L)meso-DAP(D)-NHNHBoc (1)(5.0 g), t-butylcarbonic anhydride (3.0 g)and triethylamine (2.54 g) in a mixture of water (50 ml) and dioxane (50ml) were stirred for three hours at ambient temperature. Dioxane wasdistilled off under reduced pressure and the resulting aqueous layer waswashed with ether. The aqueous layer was adjusted to pH 2 with 5%aqueous hydrochloric acid and extracted with ethyl acetate. The extractwas washed with saturated aqueous sodium chloride and then dried overanhydrous magnesium sulfate to give Z-(L)-Boc(D)meso-DAP(D)NHNHBoc(2)(6.0 g), which was recrystallized from isopropyl ether. mp 146-148(dec).

N.M.R. (CDCl₃), δ(ppm): 1.4-2.1 (6H, m), 1.67 (9H, s), 4.1-4.5 (2H, m),5.13 (2H, s), 5.50 (1H, broad s), 6.0 (1H, broad s), 7.05 (1H, broad s),7.35 (5H, s), 8.37 (1H, broad s), 8.90 (1H, broad s).

(2) Preparation 5-2

Z-(L)-Boc(D)meso-DAP(D)-NHNHBoc (2)(0.50 g) was obtained fromZ-(L)meso-DAP(D)-NHNHBoc (1)(438 mg) by substantially the same manner asthat of Example 5-1 provided that t-butylS-4,6-dimethylpyrimidine-2-ylthiocarbonate (288 mg) was employed inplace of t-butylcarbonic anhydride.

Preparation 6 ##STR166##

Z-(L)-Boc(D)meso-DAP(D)-NHNHBoc (1)(10.8 g) and N-methyl-morpholine(2.02 g) were dissolved in methylene chloride (110 ml) and stirred at-10°--15° C. Isobutyl chlorocarbonate (2.73 g) was added dropwise to thesolution and the mixture was stirred for thirty minutes at ambienttemperature. To the solution was added dropwise a solution of glycinebenzyl ester p-toluenesulfonate (6.75 g) and N-methyl morpholine (2.02g) in methylene chloride (110 ml). The solution was stirred for twohours at -10°--15° C. and for an hour at ambient temperature. Methylenechloride was distilled off under reduced pressure and the residue wasdissolved into a mixture of ethyl acetate (150 ml) and 1% aqueoushydrochloric acid (60 ml). The ethyl acetate layer was separated and wassuccessively with water 2% aqueous sodium bicarbonate and aqueous sodiumchloride in turn. The ethyl acetate layer was dried over anhydrousmagnesium sulfate and evaporated to dryness under reduced pressure. Theresidue thus obtained was recrystallized from ether to giveZ-(L)-Boc(D)-meso-DAP(L)GlyoBZl-(D)-NHNHBoc. (2)(12.3 g), mp. 85-87.

N.M.R. (CDCl₃), δ(ppm): 1.43 (18H, s), 1.5--2.2 (6H, m), 4.10 (2H, d,J=6 Hz), 4.1-4.5 (2H, m), 5.10 (2H, s), 5.17 (2H, s), 5.40 1H, d, J=8Hz), 5.90 (1H, d, J=8 Hz), 6.73 (1H, broad s), 7.33 (10H, s), 7.73 (1H,broad s), 8.4-8.6 (1H, m).

Preparation 7 ##STR167##

To a solution of di-Z-meso-DAP(L)oBzl-(D)-NHNH₂ trifluoroacetic acidsalt (1)(6.435 g) in dimethylformamide (25 ml) was added 3.29Nhydrochloric acid in dimethylformamide (5.80 ml) at -50° C. and thenisoanylnitrite (1.41 ml) was added. The solution was allowed to warm to-20° C. and then stirred for ten minutes. The resulting solution wascooled to -50° C. and triethylamine (3.97 ml) was added thereto. To thesolution was added a mixture of glycine benzyl ester p.toluensulfonate(6.41 g) and triethylamine (7.95 ml) in dimethylformamide (12 ml). Theheterogeneous mixture was stirred at 3°-7° C. for six hours and allowedto stand in a refrigerator for 48 hours. The reaction mixture wasdiluted with a mixture of ethyl acetate (170 ml) and methylene chloride(30 ml) and washed successively with 5% aqueous phosphoric acid, waterdil. sodium bicarbonate, water, and brine. The mixture was dried overanhydrous magnesium sulfate and the solvent was evaporated to dryness togive crystalline materials (6.4 g). The crystalline materials werechromatographed on silica gel (90 g) and eluted with a mixture ofmethylene chloride and ethyl acetate (3:1). The eluate was evaporated togive a residue. Crystallization was carried out from ethyl acetate togive crude crystals of di-Z-meso-DAP(L)oBzl-(D)-GlyoBzl (2)(5.32 g). mp131°-133° C.

An aliquote (450 mg) of the above crystals were further purified bychromatography on silica gel (eluted with a mixture of methylenechloride and ethyl acetate (1:3), followed by recrystallization from amixture of ethyl acetate and ether (5:2) to give fine needles. mp138°-139.5° C. [α]_(D) ²⁰ =+4.6° (C=1, chloroform).

N.M.R. (CDCl₃), δ(ppm): 1.1-2.1 (6H, m), 3.98 (2H, d, J=6 Hz), 3.8-4.5(2H, m), 5.04 (4H, s), 5.08 (2H, s), 5.10 (2H, s), 5.49 (2H, t, J=7 Hz),6.64 (1H, t, J=6 Hz), 7.28 (20H, s).

Preparation 8 ##STR168##

A solution of sodium nitrite (0.97 g) in water (10 ml) was added to atwo-phase solution of di-Z-meso-DAP(D)-NHNH₂ (6.50 g) in a mixture of 1Naqueous hydrochloric acid (42 ml) and ethyl acetate (30 ml) at 0° C. Theresulting mixture was stirred at 0° C. for five minutes and a cooledethyl acetate (30 ml) was added thereto. The ethyl acetate layer wascollected and washed four times with dil. sodium chloride, dried overanhydrous magnesium sulfate and then filtered. To the filtrate was addeda solution of H-GlyoBzl in methylene chloride (30 ml), which wasprepared by desalting glycine benzyl ester p-toluenesulfonate (11.6 g)with potassium carbonate in a mixture of methylene chloride and water ina conventional manner, and then triethylamine (1.92 ml ) was added. Theresulting mixture was kept at 4° C. for 20 hours. The reaction mixturewas washed successively with dil. hydrochloric acid and (three times),brine, dried over anhydrous magnesium sulfate and evaporated to drynessto give an oil (6.5 g), which was chromatographed on silica gel (45 g).Elution was carried out with a mixture of methylene chloride andmethanol (100:5) to give amorphous solid (5.15 g). The solid wascrystallized from a mixture of ethyl acetate and ether (1:2) to givedi-Z-meso-DAP(D)GlyoBzl (2)(1.54 g).

N.M.R. (acetone-d₆), δ(ppm): 1.3-2.4 (6H, m), 3.9-4.6 (2H, m), 4.06 (2H,d, J=6 Hz), 5.12 (4H, s), 5.18 (2H, s), 6.3-6.9 (2H, broad d, J=8 Hz),7.38 (15H, s), 7.77 (1H, t, J=6 Hz).

Preparation 9 ##STR169##

A mixture of di-Z-meso-DAP(D)-NHNHBoc (1)(8.83 g), benzyl bromide (3.66ml) and triethylamine (4.30 ml) in dimethylformamide (40 ml) was stirredat ambient temperature for 19 hours. The reaction mixture was dilutedwith ethyl acetate (150 ml) and the solution was washed successivelywith dil. phosphoric acid, water, dil. sodium bicarbonate, water andbrine. The solution was dried over anhydrous magnesium sulfate andevaporated to give an amorphous solid (9.0 g), which was purified bycolumn chromatography using silica gel (90 g), and fractions eluted witha mixture of methylene chloride and ethyl acetate (1:3) were combinedand evaporated to dryness to give a solid (8.65 g). Recrystallizationfrom a mixture of ether and hexane gave di-Z-meso-DAP(L)oBzl-(D)-NHNHBoc(2)(7.40 g).

N.M.R. (CDCl₃), δ(ppm): 1.40 (9H, s), 1.1-2.0 (6H, m), 4.0-4.7 (2H, m),5.17 (4H, s), 5.23 (2H, s), 6.87 (1H, broad s), 7.48 (15H, s), 8.57 (1H,broad s) [α]_(D) ²³ =+13.2° (C=1, chloroform).

Preparation 10 ##STR170##

To a cold mixture of di-Z-(D)meso-DAP (1)(13.8 g) and triethylamine (6.1g) in methylene chloride (150 ml) was added dropwise in the course offive minutes a cold solution of isobutoxycarbonyl chloride (5.33 g) inmethylene chloride (30 ml) at -10° C. The mixture was stirred at -10°C.--12° C. for twenty five minutes. To the mixture was added dropwise asolution of glycine ethyl ester hydrochloride (5.44 g) and triethylamine(3.94 g) in methylene chloride (200 ml) at -10° C.--12° C. in the courseof twenty minutes. The solution was stirred at -10° C. for thirtyminutes. To the reaction mixture was added water and the mixture wasadjusted to pH 2 with 1N aqueous hydrochloric acid, and then washed withsaturated aqueous sodium chloride and with 5% aqueous sodiumbicarbonate. The methylene chloride layer was washed with water and thenconcentrated. To the concentrate was added ethyl acetate and insolublematerials were filtered off and the filtrate was extracted three timeswith 5% aqueous sodium bicarbonate (pH 8). The extract was adjusted topH 2 with 1N aqueous hydrochloric acid and then extracted three timeswith ethyl acetate. The extract was washed twice with water and driedover anydrous magnesium sulfate. The extract was filtered and thefiltrate was concentrated. The oily residue thus obtained was allowed tostand in a refrigerator to give crystals. The crystals was washed withisopropyl ether and dried over anhydrous magnesium sulfate to givedi-Z-meso-DAP(OH)GlyOEt (7.73 g).

N.M.R. (DMSO-d₆), δ(ppm): 1.20 (3H, t, J=7 Hz), 1.0-1.9 (6H, m), 3.7-4.2(2H, m), 3.80 (2H, d, J=6 Hz), 4.03 (2H, q, J=7 Hz), 5.00 (4H, s), 7.33(10H, s).

Preparation 11 ##STR171##

A mixture of di-Boc-meso-DAP di-dicyclohexylamine salt (1) (18.5 g) inmethylene chloride (160 ml) was added to a solution of triethylaminehydrochloride (8.0 g) in methylene chloride (200 ml) and the mixture wasstirred at ambient temperature for two hours and cooled to -10° C. Tothe cold mixture was added dropwise a solution of ethoxycarbonylchloride (3.46 g) in methylene chloride (10 ml), maintaining thetemperature at -10° C. The solution was stirred at -10° C. for thirtyminutes. To the reaction mixture was added dropwise a mixture ofglycinethyl ester hydrochloride (4.45 g) and triethylamine (3.23 g) inmethylene chloride (200 ml) in the course of thirty minutes, maintainingthe temperature at -10° C. The mixture was stirred at the sametemperature for thirty minutes. The reaction mixture was adjusted to pH2 with 1N aqueous hydrochloric acid and then washed twice with water andtwice with saturated aqueous sodium chloride. The organic layer wasconcentrated and the concentrate was dissolved in ethyl acetate.Extraction was carried out twice with 5% aqueous sodium bicarbonate. Theextract was adjusted to pH 2 with 1N aqueous hydrochloric acid.Extraction was carried out with ethyl acetate and the organic layer waswashed successively with saturated aqueous sodium chloride, 5% aqueoussodium bicarbonate, and further washed with saturated aqueous sodiumchloride. The organic layer was dried over anhydrous magnesium sulfateand filtered. The filtrate was concentrated to give an oilydi-Boc-meso-DAP(OH)GlyOEt (2) (5.16 g).

N.M.R. (CDCl₃), δ(ppm): 1.30 (3H, t, J=7 Hz), 1.43 (18H, s), 1.1-2.1(6H, m), 4.07 (2H, t, J=6 Hz), 3.8-4.5 (2H, m), 4.15 (2H, q, J=7 Hz).

Preparation 12 ##STR172##

A solution of diazomethane (1 g/100 ml, 6 ml) was added to a solution ofdi-Z-meso-DAP-(D)-NHNHBoc (1)(0.62 g) in ether (15 ml) at 0° C. untilthe solution became yellow. The reaction mixture was evaporated todryness and the residue was chromatographed on silica gel (14 g) andelution was carried out with a mixture of methylene chloride and ethylacetate (5:3) to give di-Z-meso-DAP(L)oMe-(D)NHNHBoc.

N.M.R. (CDCl₃), δ(ppm): 1.2-2.2 (6H, m), 1.33 (9H, s), 3.73 (3H, s),4.1-4.6 (2H, m), 5.12 (4H, s), 5.74 (1H, d, J=8 Hz), 5.78 (1H, d, J=8Hz), 6.68 (1H, broad s), 7.35 (10H, s), 8.50 (1H, broad s) [α]_(D) ²³=+12.7° C. (C=1, ethanol).

Preparation 13 ##STR173##

To a suspension of Z-(L)-meso-DAP-(D)-GlyOH (1) (2.072 g) in water (20ml) was added triethylamine (2.90 ml) and the mixture was stirred at 0°C. for a few minute. A solution of t-butylcarbonic anhydride (1.54 g) indioxane (20 ml) was added to the resulting solution. The solution wasstirred at ambient temperature for 6.5 hours and concentrated. Theconcentrate was diluted with water (50 ml) and ethyl acetate (100 ml)was added thereto. The mixture was cooled to 0° C. and acidified to pH 2with 1N aqueous hydrochloric acid.

The organic layer was separated and washed with water and brine, driedover magnesium sulfate and then evaporated to giveZ-(L)-Boc(D)meso-DAP(D)GlyOH (2) (2.88 g) as an amorphous solid.

N.M.R. (DMSO-d₆), δ(ppm): 1.37 (9H, s), 1.1-1.9 (6H, m), 3.6-4.2 (2H,m), 3.73 (2H, d, J=5.5 Hz), 5.02 (2H, s), 6.75 (1H, m), 7.33 (5H, s),7.50 (1H, broad d, J=9 Hz), 8.07 (1H, broad t, J=5.5 Hz), 12.6 (2H, m).

Preparation 14 ##STR174##

Triethylamine (0.84 ml) was added to a solution ofdi-Z-meso-DAP-(D)-GlyoBzl (1)(3.0 g) in dimethylformamide (15 ml) at 0°C. and then benzyl bromide (0.72 ml) was added thereto. The mixture wasstirred at ambient temperature for 7.5 hours and triethylamine (0.42 ml)and benzyl bromide (0.36 ml) were added thereto. The mixture was stirredat ambient temperature overnight and poured into dil. phosphoric acidand then extracted with ethyl acetate (100 ml). The extract was washedsuccessively with dil. phosphoric acid, water, dil. sodium bicarbonate,dil. sodium chloride and saturated sodium chloride, and then dried overanhydrous magnesium sulfate. The solvent was evaporated to give an oil(3.4 g). The oil was chromatographed on silica gel (55 g) and elutionwas carried out with a mixture of ethyl acetate and methylene chloride(35.65 g) to give crude crystals. The crude crystals were recrystallizedfrom ethyl acetate to give di-Z-meso-DAP(L)oBzl-(D)-GlyOBzl (2) (1.38g).

N.M.R. (CDCl₃), δ(ppm): 1.1-2.1 (6H, m), 3.98 (2H, d, J=6 Hz), 3.8-4.5(2H, m), 5.04 (4H, s), 5.08 (2H, s), 5.10 (2H, s), 5.49 (2H, t, J=7 Hz),6.64 (1H, t, J=6 Hz), 7.28 (20H, s).

Preparation 15 ##STR175##

Di-Z-DAP(OH)₂ (1) (4.58 g) was dissolved in tetrahydrofuran (50 ml) andN-hydroxysuccinimide (1.15 g) was added to the solution. To theresulting solution was added a mixture of glycine benzyl esterp-toluenesulfonate (3.21 g) and triethylamine (1.4 ml) in tetrahydrofuran. N,N'-Dicyclohexylcarbodiimide (2.06 g) was added to theresulting solution under ice-cooling and the solution was stirred atambient temperature. N,N'-Dicyclohexylurea was filtered off and thefiltrate was evaporated to dryness. The residue was dissolved in ethylacetate and washed successively with dil. hydrochloric acid, saturatedsodium chloride, saturated sodium bicarbonate and saturated sodiumchloride, in turn. The organic layer was dried over magnesium sulfateand concentrated. The concentrate was chromatographed on silica gel (100g) and eluted with a mixture of chloroform and methanol (10:1). Theeluate was concentrated to dryness to give crystals ofdi-Z-DAP(OH)GlyoBzl (2) (2.67 g).

N.M.R. (CDCl₃), δ(ppm): 0.83-2.0 (6H, m), 3.3-4.5 (4H, m), 4.97 (6H,broad s), 5.0-5.6 (2H, m), 5.8-6.6 (2H, m), 7.18 (15H, m).

Preparation 16 ##STR176##

A solution of di-Z-DAP(OH)GlyoBzl (1)(5.1 g) in methanol (80 ml) washydrogenated over 5% palladium black (2 g). The reaction mixture wasfiltered and the filtrate was evaporated to give DAP(OH)GlyOH (2) (1.77g).

N.M.R. (D₂ O), δ(ppm): 1.3-2.3 (6H, m), 3.73 (1H,t, J=5.5 Hz), 3.80 (2H,s), 4.00 (1H, t, J=6 Hz).

Preparation 17 ##STR177##

To a solution of di-Boc-DAP(OH)GlyOEt (1) (6.0 g) in ethyl acetate (10ml) was was added 6N hydrochloric acid/ethyl acetate (20 ml) undercooling. The mixture was stirred under cooling for four hours to givecrystals. The crystals were collected and washed with ethyl acetate togive DAPGlyOEt-di-hydrochloric acid salt monohydrate (2) (4.18 g).

N.M.R. (D₂ O), δ(ppm): 1.30 (3H, t, J=7 Hz), 1.1-2.2 (6H, m), 3.8-4.3(2H, m), 4.11 (2H, s), 4.26 (2H, q, J=7 Hz).

Preparation 18 ##STR178##

A mixture of di-Z-meso-DAP(D)-GlyoBzl (1) (60 mg) and 10% palladiumblack (25 mg) in acetic acid (1 ml) was stirred at ambient temperatureunder hydrogen atmosphere for 4.5 hours. The reaction mixture wasfiltered through Celite. The filtrate was evaporated to givemeso-DAP(D)GlyOH (2) (39 mg).

N.M.R. (D₂ O), δ(ppm): 1.3-2.3 (6H, m), 3.73 (1H, t, J=5.5 Hz), 3.80(2H, s), 4.00 (1H, t, J=6 Hz).

Preparation 19 ##STR179##

To a solution of di-Z-meso-DAP(L)oBzl-(D)-GlyoBzl (1) (6.20 g) in aceticacid (100 ml) was added 10% palladium black (1.73 g) and the mixture wasstirred at ambient temperature under hydrogen atmosphere for twentyhours. The reaction mixture was filtered and the catalyst was washedwith acetic acid. The filtrate and the washing were combined andconcentrated. The concentrate was dissolved in a small amount of waterand the solution was evaporated to dryness. This operation was repeatedtwice. The residue was chromatographed on a macroporous non-ionicadsorption resin, HP20 (150 ml) and elution was carried out with water.The desired fraction was evaporated. The residue was dissolved in asmall amount of water and lyophilized to give amorphous solid (2.28 g).A 300 mg portion of this amorphous solid was dissolved in water (3 ml)and the solution warmed to 50° C. and then methanol was added theretountil becoming cloudy. Occasional addition of methanol was required forcompletion of crystallization. The crystals were collected by filtrationto give meso-DAP-(D)GlyOH (2) (270 mg). mp 235° C. (dec).

N.M.R. (D₂ O), δ(ppm): 1.3-2.3 (6H, m), 3.73 (1H, t, J=5.5 Hz), 3.80(2H, s), 4.00 (1H, t, J=6 Hz), [α]_(D) ²⁰ =-60.6 (C=0.507, water).

Preparation 20 ##STR180##

Z-(L)-Boc(D)-meso-DAP(L)-GlyoBzl-(D)-NHNHBoc (1) (12.0 g) washydrogenated in a mixture of methanol (100 ml) and acetic acid (2.4 ml)over 10% palladium black (2 g). After completion of the reaction, thecatalyst was removed by filtration and the filtrate was evaporated todryness under reduced pressure. To the residue was added water (30 ml)and the solution was evaporated to dryness. This operation was repeatedthree times. The residue thus obtained was triturated with ether to giveBoc(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc (2) (7.80 g). mp 130°-138° C. (dec).

N.M.R. (CD₃ OD), δ(ppm): 1.60 (9H, s), 1.63 (9H, s), 1.7-2.0 (6H, m),3.92 (2H, s), 3.8-4.1 (2H, m).

Preparation 21 ##STR181##

Di-Zmeso-DAP(OH)GlyOEt (1) (9.33 g) was hydrogenated in acetic acid (80ml) with 10% palladium black (0.93 g) under conditions of twoatmospheric pressure of hydrogen. The reaction mixture was filtered andthe filtrate was concentrated. Ethyl acetate was added to theconcentrate and-dried over magnesium sulfate to give meso-DAPGlyOEtacetic acid salt (2) (5.74 g). mp 188°-189° C. (dec.)

N.M.R. (D₂ O), δ(ppm): 1.44 (3H, t, J=7 Hz), 1.2-2.2 (6H, m), 1.93 (3H,s), 3.76 (1H, t, J=6 Hz), 3.9-4.2 (1H, m), 4.10 (2H, s), 4.20 (2H, q,J=7 Hz).

Preparation 22 ##STR182##

Boc-L-Ala-D-Glu(α-oBzl) (1) (298 mg) and anisole (0.2 ml) were added totrifluoroacetic acid (1 ml) at 0° C. and the mixture was stirred at thesame temperature for two hours. Trifluoroacetic acid was evaporated todryness and the residue was triturated with ether. The resultingcompound was dissolved in water (3 ml) and adjusted to pH 7 withtriethylamine and additional 0.1 ml of triethylamine was added to thesolution. To the resulting solution was added a solution of Lac(oAc)OSu(2) (167 mg) in dimethylformamide (3 ml) at 0° C. The resulting mixturewas stirred overnight at ambient temperature and the reaction mixturewas acidified with a diluted aqueous hydrochloric acid and thenextracted with ethyl acetate. The extract was washed with water andevaporated to dryness. The residue thus obtained was purified bypreparative thin layer chromatography using a pre-coated silica gelplate, developing with a mixture of chloroform and methanol (8:2) togive D-Lac(oAc)-L-Ala-γ-D-Glu(OH)(α-oBzl) (3) (270 mg).

N.M.R. (CD₃ OD), δ(ppm): 1.40 (3H, d, J=7 Hz), 1.47 (3H, d, J=7 Hz),2.13 (3H, s), 2.1-2.5 (4H, m), 4.4-5.3 (3H, m), 5.20 (2H, s), 7.40 (5H,s).

Preparation 23 ##STR183##

Dicyclohexylcarbodiimide (32 mg) was added to a mixture ofD-Lac(oAc)-L-Ala-D-Glu(α-oBzl)(OH) (1) (65 mg) and N-hydroxysuccinimide(18 mg) in dioxane (3 ml) at 0° C. The resulting mixture was stirredovernight at ambient temperature and filtered. The filtrate wasevaporated to dryness to give D-Lac(oAc)-L-Ala-D-Glu(α-oBzl)(oSu) (2)(80 mg).

Preparation 24 ##STR184##

Boc-L-Ala-D-Glu(α-OBzl)(OH) (1) (65 g) was dissolved in trifluoroaceticacid (260 ml) and the solution was stirred for fifteen minutes atambient temperature. The solution was evaporated under reduced pressure.The residue thus obtained was dissolved in 50% aqueous acetone (600 ml).

O-acetyl-D-lactic chloride (28.8 g) was added dropwise to the solutionwhile stirring under ice-cooling, maintaining the pH 7-8.

The stirring was continued for an hour at the same temperature and thenacetone was evaporated under reduced pressure. The resulting aqueoussolution was washed with ethyl acetate (300 ml) and adjusted to pH 2with 10% aqueous hydrochloric acid. Extraction was carried out twicewith ethyl acetate (600 ml and 300 ml). The ethyl acetate layer waswashed with saturated aqueous sodium chloride solution and dried overanhydrous magnesium sulfate and then evaporated under reduced pressureto give D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)(OH) (2) (64.7 g).

N.M.R. (CDCl₃), δppm: 1.40 (3H, d, J=7 Hz), 1.47 (3H, d, J=7 Hz), 2.13(5H, s), 2.1-2.5 (4H, m), 4.4-5.3 (3H, m), 5.20 (2H, s), 7.40 (5H, s).

Preparation 25 ##STR185##

Z-(L)-Boc-(D)meso-DAP-(D)GlyOH (1) (2.80 g) was hydrogenated in methanol(56 ml) over 10% palladium black (0.82 g) under conditions of ordinaryatmospheric pressure of hydrogen at ambient temperature for four hours.To the reaction mixture was added methanol (50 ml) and the resultingmixture was warmed to 40° C. and then filtered. The catalyst was washedwell with methanol and filtered. The filtrate and washings were combinedand concentrated to give crystals. The crystals was diluted withisopropanol and cooled in a refrigerator and then washed with ether togive Boc-(D)meso-DAP(D)GlyOH (2) (1.687 g)

N.M.R. (CD₃ OD+D₂ O), δ(ppm): 1.3-2.2 (6H, m), 1.40 (9H, s), 3.66 (1H,t, J=5 Hz), 3.7-4.2 (1H, m), 3.88 (2H, s) [α]_(D) ²³ =+12.2°(C=0.5,methanol).

Preparation 26 ##STR186##

To a solution of Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyoBzl (1) (105mg) in methanol was added 0.1N aqueous sodium hydroxide (1.5 ml). Themixture was stirred at ambient temperature for two hours and evaporated.The resulting aqueous solution was acidified to pH 2 with 5% aqueoushydrochloric acid and extracted with ethyl acetate.

The extract was washed with water, dried over magnesium sulfate andevaporated to give Z-(L)-Boc-(D)-mesoDAP(D)-NHNHBoc-(L)-GlyOH (2) (85mg).

N.M.R. (CDCl₃), δ(ppm): 1.43 (18H), s), 1.3-2.1 (6H, m), 3.9-4.5 (4H,m), 5.10 (2H, s), 6.2-6.4 (4H, m), 7.10 (1H, broad s), 7.33 (5H, s),8.95 (1H, broad s).

Preparation 27 ##STR187##

Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyoBzl (1) (2.0 g) was added totrifluoroacetic acid (10 ml) and the mixture was stirred at ambienttemperature for 15 minutes. Trifluoroacetic acid was distilled off andthe residue was dissolved in 50% aqueous dioxane (30 ml) and then cooledto 0° C. To this solution was added N-bromosuccinimide (1.04 g) and themixture was stirred at the same temperature for an hour. Dioxane wasdistilled off and the residue was adjusted to pH 6 with saturatedaqueous sodium bicarbonate. The precipitated crystalline solid wasfiltered and washed with water to give Z-(L)-mesoDAP-(L)-GlyoBzl (2)(1.15 g).

N.M.R. (DMSO-d₆), δ(ppm): 1.2-2.0 (6H, m), 3.8-4.2 (4H, m), 5.05 (2H,s), 5.15 (2H, s), 7.40 (10H, s).

Preparation 28 ##STR188##

A solution of Z-(L)-mesoDAP-(L)GlyoBzl (1) (100 mg) in a mixture ofmethanol (10 ml) and water (3 ml) was hydrogenated over 10%palladium-black (50 mg) under an ordinary atmospheric pressure ofhydrogen. The reaction mixture was filtered and the filtrate wasevaporated. The residue was pulvelized to give mesoDAP-(L)GlyOH (2) (44mg).

N.M.R. (D₂ O), δ (ppm): 1.3-2.1 (6H, m), 3.73 (1H, t, J=6 Hz), 3.80 (2H,s), 4.01 (1H, t, J=6 Hz).

Preparation 29 ##STR189##

P-Toluenesulfonic acid (monohydrate) (0.56 g) was added to a solution ofBoc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyoH (1) (1.39 g). To the mixture wasadded a solution of diphenyldiazomethane (0.62 g) in methanol (1 ml).The resulting mixture was stirred at ambient temperature for 30 minutes.

To the reaction mixture was added an additional diphenyldiazomethane(0.78 g) until the starting material (1) was disappeared on thin layerchromatography.

An excess of the reagent was destroyed by adding acetic acid and themixture was adjusted to pH 8 with saturate aqueous sodium bicarbonateand then evaporated. The residue was dissolved in ether (3 ml) andtriturated with n-hexane (5 ml). The solvents were removed bydecantation. This operation was further repeated twice. The residue wasput on a column of silica-gel (30 g) and eluted with a mixture of ethylacetate and methanol (10:1).

The fractions containing the object compound (2) were combined andevaporated to give a solid, which was purified by reprecipitation from amixture of ether and n-hexane (I:2) to giveBoc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyoBzh(2)(1.22 g).

N.M.R. (CDCl₃), δ(ppm): 1.33 (18H, s), 1.1-1.8 (6H, m), 3.1-3.5 (1H, m),3.9-4.3 (3H, m), 5.2-5.5 (1H, m), 6.75 (1H, s), 7.3 (10H, s), 7.6-8.0(1H, m).

Preparation 30 ##STR190##

To a mixture of Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (1) (1.40 g) andglycine methyl ester hydrochloride (392 mg) in dimethyl formamide (10ml) were added N-hydroxysuccinimide (0.45 g) anddicyclohexylcarbodiimide (0.645 g) at 0° C., and the resulting mixturewas stirred at the same temperature for 30 minutes and at ambienttemperature for overnight. The resulting precipitate was filtered offand the filtrate was concentrated. The concentrate was dissolved inethyl acetate (50 ml) and the solution was washed successively with 0.5Nhydrochloric acid, water, 2.5% sodium bicarbonate and water.

The organic layer was dried over magnesium sulfate and evaporated togive Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)GlyoMe (2)(1.45 g).

N.M.R. (CDCl₃), δ(ppm): 1.40 (18H, s), 1.5-2.0 (6H, m), 3.70 (3H, s),3.98 (2H, d, J=6 Hz), 4.0-4.5 (2H, m), 5.07 (2H, s), 5.45 (1H, d, J=8Hz), 5.93 (1H, d, J=8 Hz), 6.83 (1H, broad s), 7.1-7.3 (1H, m), 7.33(5H, s), 8.63 (1H, broad s).

Preparation 31 ##STR191##

To a solution of Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOMe (1)(1.41g) in a mixture of water (14 ml) and methanol (10 ml) was added 1Nsodium hydroxide (2.3 ml) at 0° C.

The mixture was stirred at the same temperature for 30 minutes and atambient temperature for 3.5 hours. Methanol was distilled off and theresulting solution was washed with ethyl acetate. The aqueous layer wasadjusted to pH 3 with 5% hydrochloric acid and extracted with ethylacetate.

The extract was washed with a saturated sodium chloride, dried overmagnesium sulfate and then evaporated to giveZ-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (2)(1.24 g).

N.M.R. (CDCl₃), δ(ppm): 1.43 (18H, s), 1.3-2.1 (6H, m), 3.9-4.5 (4H, m),5.10 (2H, s), 6.2-6.4 (4H, m), 7.10 (1H, broad s), 7.33 (5H, s), 8.95(1H, broad s).

Preparation 32 ##STR192##

Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1)(270 mg) was added totrifluoroacetic acid (5 ml) and the mixture was stirred at ambienttemperature for 15 minutes.

Trifluoroacetic acid was distilled off and the residue was pulverizedwith ether. The solvent was removed by decantation and the solidmaterial thus obtained was dissolved in 60% aqueous acetic acid (30 ml)and manganese dioxide (180 mg) was added to the solution. The resultingmixture was stirred at ambient temperature for an hour. The reactionmixture was filtered and the filtrate was evaporated. The residue wasdissolved in water and the solution was evaporated.

The residue was dissolved in 50% ethanol (60 ml) and adjusted to pH 9with dil. aqueous ammonia and stored in a refrigerator. The solution wastreated with an active carbon (30 mg) and filtered. The filtrate wasconcentrated and the concentrate was dissolved in water (20 ml) and puton a column of carbon (15 ml).

The column was washed with water and eluted with 70% aqueous acetone.The extract was evaporated to give Z-(L)-mesoDAP-(L)-GlyOH (2)(85 mg).

M.p. 210° C.(dec.).

I.R. (Nujol): 3300, 2600-2400 (shoulder), 1710 (shoulder), 1680, 1640cm⁻¹.

N.M.R. (DMSO-d₆), δ(ppm): 1.10-2.20 (6H, m), 3.30 (1H, m), 3.65 (2H, d,J-7 Hz), 4.00 (1H, m), 5.00 (2H, s), 7.30 (5H, s).

Preparation 33 ##STR193##

A solution of di-Z-meso-DAP(L)oBzl-(D)-NHNHBoc (1) (6.68 g) intrifluoroacetic acid (30 ml) was stirred at ambient temperature fortwenty minutes. The resulting solution was concentrated and the residuewas dissolved in methylenechloride. The solution was evaporated underreduced pressure and the residue was crystallized from ether to givedi-Z-meso-DAP(L)oBzl-(D)-NHNH₂ trifluoroacetic acid salt. (2) (6.627 g).mp 113°-114° C.

N.M.R. (DmSo-d₆) δ(ppm): 1.1-2.0 (6H, m), 3.6-4.3 (2H, m), 5.03 (4H, s),5.11 (2H, s), 7.33 (15H, s), 7.1-8.4 (6H, m).

Preparation 34 ##STR194##

A mixture of di-Z-meso-DAP(D)-NHNHBoc (1) (6.41 g) and anisole (3 ml) intrifluoroacetic acid (17 ml) was stirred at 0° C. for 1.5 hours. Thereaction mixture was concentrated under reducing pressure and ether (50ml) was added to the concentrate. The ether solution was triturated togive precipitates. The precipitates were washed twice with ether andpumped to give di-Z-meso-DAP(D)NHNH₂ trifluoroacetic acid salt.(2) (7.0g).

N.M.R. (CD₃ OD) δ(ppm): 1.2-2.1 (6H, m), 3.9-4.5 (2H, m), 5.05 (4H, s),7.25 (10H, s).

Preparation 35 ##STR195##

To a mixture of di-Z-L-DAP (1)(4.59 g) and N-methylmorpholine (2.2 ml)in methylene chloride (50 ml) was added iso-butoxycarbonyl chloride(1.69 ml) at -20° C. and the mixture was stirred at -10° C.--15° C. for30 minutes. The reaction mixture was cooled to -30° C. and a mixture ofH-GlyoBzl p-toluenesulfonate (4.38 g) and N-methylmorpholine (1.43 ml)in methylene chloride (25 ml) was added thereto. The mixture was stirredat -10° C. for an hour and then allowed to warm to ambient temperature.The reaction mixture was concentrated and the concentrate was pouredinto a mixture of ethyl acetate and dil. hydrochloric acid. Insolublematerials were filtered off and the organic layer was separated andwashed successively with dil. sodium bicarbonate, dil. hydrochloricacid, water and brine. During this operation, crystals were separatedout which was collected by filtration to give di-Z-L-DAP-mono-GlyoBzl(2)(0.79g). The filtrate was dried over magnesium sulfate andevaporated. The residue was combined with the insoluble materialsdescribed above and then recrystallized from ethyl acetate to giveadditional di-Z-L-DAP-mono-GlyoBzl (2)(2.55 g).

N.M.R. (DmSo-d₆): 1.1-1.9 (6H, m), 3.89 (2H, broad d, J=5 Hz), 3.7-4.4(2H, m), 5.00 (4H, s), 5.10 (2H, s), 7.1-7.6 (2H, m),7.32 (15H, s), 8.31(1H, t, J=5 Hz).

Preparation 36 ##STR196##

Di-Z-L-DAP-mono-GlyoBzl (1)(3.12 g) was dissolved in a mixture ofmethanol (10 ml) and acetic acid (60 ml) by warming. To the solution wasadded 10% palladium black (850 mg) and the mixture was stirred under anatmospheric pressure of hydrogen overnight, during which time an whiteprecipitate was appeared. The catalyst and the precipitate werecollected by filtration and the filter cake was washed with 50% aqueousacetic acid and water. The filtrate and the washings were combined andevaporated. The residue was dissolved in water and the solution wasevaporated in order to remove acetic acid. This operation was repeatedtwice and the residue was triturated with hot water and then methanolwas added thereto. The suspension was cooled in a refrigerator and theresulting precipitate was filtered and washed with methanol and ether togive L-DAP-monoGlyOH (2)(1.295 g).

I.R. (Nujol): 1660, 1640 (shoulder), 1590 (broad), 1530 cm⁻¹.

N.M.R. (D₂ O) δ(ppm): 1.2-2.3 (6H, m), 3.5-4.3 (2H, m), 3.87 (2H, s).

Preparation 37 ##STR197##

To a mixture of L-DAP-monoGlyOH (1)(1.25 g) and cupric chloride(dihydrate)(1)(0.86 g) in water (120 ml) were added 3N sodium hydroxide(5.5 ml) and carbobenzyloxy chloride (3.6 ml) at 5° C. The mixture wasstirred at 5° C. for 4.25 hours, during which time the pH of thereaction mixture was kept at 11-11.5 by adding 3N sodium hydroxide.

To the reaction mixture was added an additional carbobenzyloxy chloride(1.08 ml) and the resulting mixture was stirred at the same temperaturefor 6 hours, maintaining pH 11-11.5 with 3N sodium hydroxide. Anotherportion of carbobenzyloxy chloride (1.08 ml) was added to the reactionmixture and the resulting mixture was stirred at 5° C. for 2.5 hours,keeping the pH at 11-11.5. The reaction mixture was acidified to pH 2with dil. hydrochloric acid and washed twice with ether. The aqueouslayer was adjusted to pH 4.5 with dil. sodium hydrochloride and thenhydrogen sulfide gas was bubbled at 5° C. The resulting precipitate wasfiltered off and the filtrate was concentrated to 50 ml. The concentratewas adjusted to pH 2 and chromatographed on a column of a macroporousnon-ionic adsorption resin, HP 20 (70 ml). Fractions eluted with 50%aqueous methanol were collected and evaporated. The residue wastriturated with ether containing a small amount of methanol to giveZ-(α)-L-DAP-(ε)-GlyOH (2)(1.30 g).

N.M.R. (D₂ O), δ(ppm): 1.2-2.2 (6H, m), 3.88 (2H, d, J=2.5 Hz), 3.8-4.3(2H, m), 5.10 (2H, s), 7.43 (5H, s).

Preparation 38

(1) Step 1 ##STR198##

D-Glu(α-oBzl)(1)(4.74 g) was dissolved in a mixture of dioxane (60 ml)and water (60 ml) containing triethylamine (2.02 g). To this solutionwas added Boc-Gly-N-Su (2)(5.16 g), and the mixture was allowed to standovernight at ambient temperature. The resulting reaction mixture wasevaporated to give an oily residue which was dissolved in ethyl acetateand then washed with water. The organic layer was dried over magnesiumsulfate and evaporated to give an oily residue (5.40 g). Analyticalsample of the compound (3) was purified as dicyclohexylamine salt whichwas recrystallized from ethyl acetate-diisopropyl ether to givedicyclohexylamine salt of Boc-Gly-D-Glu(α-oBzl). mp 127°-129° C.

N.M.R. (CDCl₃), δ(ppm): 1.40 (9H, s), 0.84-2.40 (24H, m), 2.92 (2H, m),3.80 (2H, d, J=7 Hz), 4.08 (1H, q, J=7 Hz), 4.48 (1H, m), 5.12 (2H, s),5.64 (1H, m), 7.32 (5H, s), 8.54 (1H, d, J=7 Hz), 9.18 (1H, s).

(2) Step 2 ##STR199##

Boc-Gly-D-Glu(α-oBzl)(3)(4.30 g) was dissolved in trifluoroacetic acid(20 ml) and the solution was kept at ambient temperature for 30 minutes.Evaporation of the solvent under reduced pressure gave an oily residue,to which toluene was added and evaporated in vacuo. The resulting oilyresidue was dissolved in 50% aqueous acetone (20 ml) and the solutionwas adjusted to pH 7.0-8.0 by adding saturated sodium bicarbonatesolution. To this stirred solution cooled in an ice-bath, was addeddropwise acetyl-D-lactyl chloride (1.97 g) and the mixture was stirredat the same temperature for an hour. The reaction mixture was extractedwith ethyl acetate and the aqueous layer was treated withconc.hydrochloric acid to adjust the pH to 3.0. The aqueous layer wasextracted with ethyl acetate and the organic layer was washed withwater, dried over magnesium sulfate and evaporated to give an oilyresidue (2.8 g). The residue was purified as dicyclohexylamine saltwhich was recrystallized from ethyl acetate-diisopropyl ether to giveD-Lac(oAc)-Gly-D-Glu(α-oBzl) (4)(2.1 g). mp 117°-118° C.

N.M.R. (CDCl₃), δ(ppm): 0.80-2.52 (24H, m), 1.48 (3H, d, J=7 Hz), 2.14(3H, s), 2.96 (2H, m), 3.96 (2H, d, J=7 Hz), 4.46 (1H, q, J=7 Hz), 5.16(2H, s), 5.20 (1H, q, J=7 Hz), 7.32 (5H, s), 8.92-9.28 (3H, m).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Preparation 38.

Preparation 39

(1) Step 1

Dicyclohexylamine salt of Boc-L-Val-D-Glu(α-oBzl) mp. 130° C.

N.M.R. (CDCl₃), δ(ppm): 0.91 (3H, d, J=7 Hz), 0.94 (3H, d, J=7 Hz),1.0-2.60 (25H, m), 2.96 (2H), 4.00 (1H, m), 4.44 (1H, m), 5.14 (2H, s),5.36 (1H, m), 7.24 (1H, m), 7.32 (5H, s), 8.54 (1H), 9.38 (1H, s).

(2) Step 2

Dicyclohexylamine salt of D-Lac(oAc)-L-Val-D-Glu (α-oBzl). mp. 121°-122°C.

N.M.R. (CDCl₃), δ(ppm): 0.91 (3H, d, J=7 Hz), 0.94 (3H, d, J=7 Hz),1.00-2.60 (25H, m), 1.46 (3H, d, J=7 Hz), 2.14 (3H, s), 3.00 (2H, m),4.28-4.60 (2H, m), 5.16 (2H, s), 5.20, 7.00 (1H, d, J=7 Hz), 7.32 (5H,s), 9.04 (1H, d, J=7 Hz), 9.28 (1H, s).

Preparation 40

(1) Step 1

Boc-L-Ser(oBzl)-D-Glu(α-oBzl). mp 84°-85° C.

N.M.R. (CDCl₃), δ(ppm): 1.44 (9H, s), 1.76-2.48 (4H, m), 3.60,3.65 (2H,doublet of AB quartet, J=4 Hz, 7 Hz), 4.36 (1H), 4.52 (2H, s), 4.68(1H), 5.16 (2H, s), 5.56 (1H), 7.28 (5H, s), 7.32 (5H, s), 7.04-7.40(1H, m), 9.62 (1H, broad s).

(2) Step 2

Dicyclohexylamine salt of D-Lac(oAc)-L-Ser(oBzl)-D-Glu(α-oBzl). mp124°-125° C.

N.M.R. (CDCl₃), δ(ppm): 1.00-2.40 (24H, m), 1.46 (3H, d, J=7 Hz), 2.12(3H, s), 2.92 (2H, m), 3.62, 3.88 (2H, doublet of AB quartet, J=4 Hz, 10Hz), 4.40-4.72 (2H, m), 4.52 (2H, s), 5.16 (2H, s), 5.17 (1H, q, J=7Hz), 7.12 (1H, d, J=7 Hz), 7.28 (5H, s), 7.32 (5H, s), 8.44 (1H, d, J=7Hz), 8.56 (1H, broad s).

Preparation 41

(1) Step 1

Boc-L-Phe-D-Glu(α-oBzl). mp 131° C.

N.M.R. (DMSO-d₆), δ(ppm): 1.30 (9H, s), 1.60-2.33 (4H, m), 2.67-3.0 (2H,m), 3.90-4.66 (2H, m), 5.13 (2H, s), 7.21 (5H, s), 7.33 (5H, s), 8.30(1H, d, J=7 Hz).

(2) Step 2

D-Lac(oAc)-L-Phe-D-Glu(α-oBzl). mp 148°-150° C. (dec.)

N.M.R. (DMSO-d₆), δ(ppm): 1.13 (3H, d, J=7 Hz), 2.00 (3H, s), 1.80-2.40(4H, m), 2.67-3.30 (2H, m), 4.20-4.83 (2H, m), 4.91 (1H, q, 7 Hz), 5.17(2H, s), 7.23 (5H, s), 7.36 (5H, s), 8.25 (2H, t, J=7 Hz).

Preparation 42 ##STR200##

To a solution of L-Ala-D-Glu(α-oBzl) (1) hydrochloric acid salt (5.40 g)in 50% aqueous dioxane (100 ml) were added triethylamine (3.16 g) andBoc-D-AlaoSu (4.48 g), and the mixture was stirred overnight at ambienttemperature. After evaporation of dioxane, the aqueous layer wasacidified to pH 1 with dil hydrochloric acid and extracted with ethylacetate. The extract was washed with water, dried over magnesium sulfateand evaporated. The residue was crystallized from a mixture of ethylacetate and ether and filtered to give Boc-D-Ala-L-Ala-D-Glu(α-oBzl)(3)(6.90 g).

N.M.R. (acetone-d₆), δ(ppm): 1.32 (6H, d, J=7 Hz), 1.40 (9H, s),2.09-2.54 (4H, m), 3.88-4.72 (3H, m), 5.15 (2H, s), 6.17 (1H, d, J=7Hz), 7.37 (5H, s), 7.34-7.67 (2H, m).

Preparation 43

(1) Step 1 ##STR201##

To a solution of Z-(L)-Boc-(D)-mesoDAP (D)-NHNHBoc (2) (1.50 g.) andN-methylmorpholine (0.32 ml.) in methylenechloride (15 ml.) was addedisobutyl chlorocarbonate (0.375 ml.) at -25° C. After stirring at -15°C. for 30 minutes, the solution was cooled to -45° C. To the resultingsolution were added a solution of B-AlaOBzl,. P-toluenesulfonate (1.075g.) and N-methylmorpholine (0.34 ml.) inmethylenechloride-dimethylformamide (10 ml.-1 ml.). The reaction mixturewas stirred at -15° C--10° C. for an hour, allowed to warm to ambienttemperature and then concentrated. The concentrate was taken up intoethyl acetate (600 ml.) and washed in turn with dil hydrochloric acid,water (×2), dil sodiumbicarbonate, water and brine. The concentrate wasdried over magnesium sulfate and the solvent was distilled off to giveZ-(L)-Boc-(D)-meso DAP-(L)-β-AlaOBzl-(D)-NHNHBoc (2) (2.00 g.).

N.M.R. (DMSO-d₆), δ(ppm): 1.1-1.9 (6H, m), 3.1-3.5 (2H, m), 3.7-4.1 (2H,m), 4.98 (2H, s), 5.06 (2H, s), 6.5-6.8 (1H, m), 7.3 (1H, m), 7.95 (1H,m), 8.66 (1H, broad s), 9.53 (1H, broad s).

(2) Step 2 ##STR202##

A suspension of 10% of palladium-carbon (0.50 g.) in water (5 ml.) wasadded to a solution of compound (2) (1.95 g.) in methanol (40 ml.). Themixture was stirred under hydrogen atmosphere. The reaction mixture wasfiltered and the filtrate was concentrated. Water was coevaporated withethanol to give an amorphous solid. Trituration with ether andfiltration gave Boc (D) mesoDAP-(L)-β-AlaOH-(D)-NHNHBoc (3) (1.25 g.).

N.M.R. (D₂ O), δ(ppm): 1.0-2.2 (6H, m), 1.45 (18H, s), 2.41 (2H, t, J=7Hz), 3.47 (2H, t, J=7 Hz), 3.7-4.2 (2H, m).

The following compounds were prepared in substantially the same manneras steps 1 and 2 of Preparation 43.

Preparation 44

(2) Step 1 ##STR203##

N.M.R. (DMSO-d₆), δ(ppm): 1.1-2.5 (10H, m), 1.35 (18H, s), 3.5-4.6 (3H,m), 4.97 (2H, s), 5.01 (2H, s), 5.07 (2H, s), 6.6-6.8 (1H, m), 7.30(15H, s), 7.7-8.8 (2H, m).

(2) Step 2 ##STR204##

N.M.R. (D₂ O), δ(ppm): 0.6-2.6 (28H, m), 3.5-4.4 (3H, m).

Preparation 15

(1) Step 1 ##STR205##

N.M.R. (DMSO-d₆), δ(ppm): 1.33 (18H, s), 1.25-2.00 (9H, m), 3.66-4.50(3H, m), 5.02 (2H, s), 5.11 (2H, s), 7.33 (10H, s).

(2) Step 2 ##STR206##

N.M.R. (D₂ O), δ(ppm): 1.50 (18H, s), 1.15-2.15 (9H, m), 3.83-4.33 (3H,m).

Preparation 46

(1) Step 1 ##STR207##

I.R. (Nujol): 3270, 1695 (broad), 1640 cm⁻¹.

(2) Step 2 ##STR208##

N.M.R. (D₂ O), δ(ppm): 1.2-2.2 (6H, m), 1.41 (18H, s), 2.93 and 3.10(3H, a pair of singlets), 3.5-4.5 (4H, m).

Preparation 47

(1) Step 1 ##STR209##

N.M.R. (DMSO), δ(ppm): 1.0-1.9 (6H, m), 1.37 (18H, s), 2.8-3.2 (2H, m),3.5-4.3 (2H, m), 4.3-4.8 (1H, m), 4.99 (2H, s), 5.08 (2H, s), 6.4-6.8(1H, m), 7.1 (1H, m), 7.15 (5H, s), 7.28 (10H, m), 8.28 (1H, broad d,J=7 Hz), 8.4-8.8 (1H, m), 9.50 (1H, broad s).

(2) Step 2 ##STR210##

N.M.R. (D₂ O), δ(ppm): 0.8-2.0 (6H, m), 1.53 (18H, s), 2.5-4.2 (5H, m),b 7.41 (5H, s).

Preparation 48

(1) Step 1 ##STR211##

N.M.R. (DMSO-d₆), (ppm): 1.0-2.0 (9H, m) 1.42 (18H, s), 3.7-4.7 (3H, m),5.05 (2H, s), 5.15 (2H, s), 6.6-7.0 (1H, s), 7.1-7.8 (1H, m), 7.38 (10H,s), 8.40 (1H, broad s, J-7 Hz), 8.5-8.9 (1H, m), 9.65 (1H, broad s).

(2) Step 2 ##STR212##

N.M.R. (CD₃ OD-D₂ O), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.44 (9H, s), 1.46(9H, s), 1.1-2.2 (6H, m), 3.8-4.3 (3H, m).

Preparation 49 ##STR213##

To a cooled solution of di-Z-mesoDAP (1) (3.50 g) in methylene chloride(70 ml.) was added phosphorus pentachloride (3.50 g.) at 0° C., and themixture was stirred at the same temperature for 15 minutes. Thetemperature was raised to ambient temperature and then the mixture wasstirred for 15 minutes. The reaction mixture was then warmed to 50° C.and stirred for 20 minutes. After standing for 20 minutes at 0° C., theresulting crystalline solid was filtered and washed with methylenechloride to give mesoDAP-N-carboxylic anhydride (2) (1.68 g.).

I.R. (Nujol): 3250, 1840, 1765 cm⁻¹.

N.M.R. (DMSO-d₆), δ(ppm): 1.2 2.0 (6H, m), 4.43 (2H, t, J=5 Hz), 9.06(2H, s).

Preparation 50 ##STR214##

To a cooled mixture of t-butylcarbazate (1.58 g.) and oxalic acidhydrate (1.26 g.) in methanol (7 ml.) was added a solution of mesoDAPN-carboxylic anhydride (1.23 g.) in methyl cyanide (7 ml.). Afterstirring at 0° C. for 10 minutes, methyl cyanide (7 ml.) was added tothe solution and the mixture was stirred at the same temperature for 30minutes. The resulting crystalline solid was filtered and washed withmethyl cyanide to give mesoDAP-di NHNHBoc oxalate (2) (2.66 g.).

I.R. (Nujol); 3600-2200, 1720, 1680, 1610 cm⁻¹.

N.M.R. (D₂ O), δ(ppm): 1.47 (18H, s), 1.3 2.3 (6H, m), 4.10 (2H, t, J=6Hz).

Preparation 51 ##STR215##

MesoDAP di-NHNH Boc acetate (2) was prepared from mesoDAP N-carboxylicanhydride (1) in substantially the same manner as that of Preparation50.

N.M.R. (D₂ O), δ(ppm): 1.52 (18H, s), 1.95 (6H, s), 1.4-2.3 (6H, m),4.08 (2H, t, J=7 Hz).

Preparation 52

(1) Step 1 ##STR216##

Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (1) (2.26 g) was added totrifluoroacetic acid (6 ml) and stirred for 15 minutes at roomtemperature. After evaporation of trifluoroacetic acid, the resultingoily residue was dissolved in 0.1N sulfuric acid (42 ml) and cooled inan ice-bath. To this solution was added a solution of sodium periodate(1.078 g) in water (15 ml) and the mixture was stirred for 1 hour at thesame temperature. The reaction mixture was treated with an aqueoussolution of sodium bisulfite until the dark color was clear. Afteradjusting the pH of the solution to 7.0, the solution was concentratedto about 10 ml and the pH was adjusted to 2.0. This solution was appliedto a column of a macroporous nonionic adsorption resin, HP-20 (100 ml),which was eluted with water and then with water-methanol (3:2).Evaporation of the latter fractions gave Z-(L)-mesoDAP (2) (1.0 g) as awhite crystalline residue.

m.p. ˜245° C. (dec)

I.R. (Nujol): 3500, 3350, 3200, ˜2600 (broad), 1690 cm⁻¹.

N.M.R. (DMSO-d₆): δ(ppm): 1.16-2.0 (6H, m), 3.30 (1H, broad signal),3.90 (1H, broad signal), 5.00 (2H, s), 7.33 (5H, s).

(2) Step 2 ##STR217##

To a mixture of Z-(L)-mesoDAP (2) (1.50 g) and triethylamine (1.0 g) inaqueous dioxane (dioxane 30 ml and water 20 ml) was added Boc-D-Ala-OSu(3) (1.32 g) and the mixture was allowed to stand overnight at roomtemperature. Dioxane was evaporated, and the resulting aqueous solutionwas acidified with diluted hydrochloric acid and extracted with ethylacetate. The extract was washed with water, dried over magnesium sulfateand evaporated to give Boc-D-Ala-(D)-Z-(L)-mesoDAP (4) (1.90 g) as awhite foam.

I.R. (Nujol): 3300, 1700 (broad) cm⁻¹.

N.M.R. (DMSO-d₆), δ(ppm): 1.20-2.0 (18H, m), 3.70-4.40 (3H, m), 5.00(2H, s), 7.37 (5H, s).

(3) Step 3 ##STR218##

A solution of Boc-D-Ala-(d)-Z-(L)-mesoDAP (4.1 g.) in acetic acid (40ml.) was hydrogenated over 10% palladium-charcoal (0.40 g.). Thecatalyst was removed by filtration and the filtrate was evaporated togive a pasty residue, to which toluene was added and evaporated to giveBoc-D-Ala-(D)-mesoDAP (5) (1.50 g.) as a white powder.

I.R. (Nujol): 3300, 2600-2400 (broad), 1720 1600 (broad) cm⁻¹.

N.M.R. (D₂ O), δ(ppm): 1.17-2.0 (18H, m), 3.30 (1H, broad signal),3.80-4.30 (2H, m).

Preparation 53

(1) Step 1 ##STR219##

To a suspension of D-alanine (2.89 g) in a mixture of methylene chloride(30 ml) and dimethylformamide (12 ml) was addedbis(trimethylsilyl)acetamide (30 ml) and the mixture was stirred for 15hours at room temperature to give a clear solution. To a solution of themixed anhydride of Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (1) (13.45 g) inmethylene chloride (140 ml), prepared by adding N-methylmorphorine (3.03g) and isobutyl chloroformate (4.10 g) at -15° and stirring the mixturefor 40 minutes at the same temperature, was added in one portion theabove solution of D-alanine trimethylsilyl ester at -40° C. Then thereaction mixture was allowed to warm up to 0° C. and stirred for 2hours. The reaction mixture was evaporated and the residue was dissolvedin water, acidified with diluted hydrochloric acid and extracted withethyl acetate. The extract was washed with water, dried over magnesiumsulfate and concentrated to give a white powder (12.25 g) ofZ-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (2).

I.R. (Nujol): 3300, 2600-2400(broad), 1720-1640(broad) cm⁻¹.

NMR (DMSO-d₆), δ(ppm): 1.20-2.00(27H, m), 3.70-4.40 (3H, m), 5.00(2H,s), 7.36(5H, s).

(2) Step 2 ##STR220##

Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (2) (14.60 g) wasdissolved in trifluoroacetic acid (20 ml) and stirred for 15 minutes atroom temperature. Trifluoroacetic acid was removed by evaporation andthe residual oil was dissolved in a mixture of 1N sulfuric acid (25 ml)and water (30 ml). The resulting solution was treated with a solution ofsodium periodate (5.60 g) in water (20 ml) under cooling in an ice-bathand kept for 1 hour at the same temperature. The reaction mixture wasthen treated with an aqueous sodium bisulfite solution until the colorof the solution became clear. This solution was applied to a column ofHP-20, an macroporus non-ionic adsorption resin (300 ml), which waseluted successively with water and methanol-water (1:2). Evaporation ofthe latter fractions gave a crystalline residue (6.80 g) ofZ-(L)-mesoDAP-(L)-D-AlaOH (3).

m.p. ˜150° C. (dec).

I.R. (Nujol): 3300, 2600-2400(broad), 1720(shoulder), 1700, 1640 cm⁻¹.

NMR (DMSO-d₆), δ(ppm): 1.30 (3H, d, J=7 Hz), 1.10-2.30 (6H, m), 3.43(1H, broad s), 3.80-4.50 (2H, m), 5.10 (2H, s), 7.40 (5H, s).

(3) Step 3 ##STR221##

To a mixture of Z-(L)-mesoDAP-(L)-D-AlaOH (3) (5.70 g) and triethylamine(4.50 g) in water (50 ml) was added a solution2-t-butoxycarbonyloxyimino-2-phenylacetonitrile (3.55 g) in aceton (50ml) and the mixture was stirred for 1 hour at room temperature. Afterstanding overnight at the same temperature, the reaction mixture wasconcentrated and the resulting aqueous solution was washed with ethylacetate. The aqueous layer was acidified with 1N hydrochloric acid andextracted with ethyl acetate. The extract was washed with water, driedover magnesium sulfate and evaporated to give a white foam (6.0 g) ofZ-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (4).

I.R. (Nujol): 3300, 2600-2400, 1720-1620 (broad) cm⁻¹.

NMR (DMSO-d₆), δ(ppm): 1.30-2.0 (18H, m), 3.70-4.3 (3H, m), b 5.10 (2H,s), 7.40 (5H, s).

(4) Step 4 ##STR222##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (4) (5.80 g) was dissolved in aceticacid (60 ml) and hydrogenated over 10% palladium-charcoal (0.50 g). Thecatalyst was removed by filtration and the filtrate was evaporated togive an oily residue, which was dissolved in water (30 ml) and appliedto a column of HP-20 (150 ml), a macroporous non-ionic adsorption resin.The column was eluted successively with water and methanol-water (3:7).The latter fractions were combined and evaporated to gave a white powder(3.80 g) of Boc-(D)-mesoDAP-(L)-D-AlaOH (5).

I.R. (Nujol): 3250, 2600-2400 (broad), 1720 (shoulder), 1670 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.20-2.20 (18H, m), 4.00 (2H, t, J=7 Hz), 4.30 (1H,q, J=7 Hz).

Preparation 54

(1) Step 1 ##STR223##

Z-(L)-mesoDAP-(L)-GlyOH(2) was prepared fromZ-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(1) in substantially the samemanner as that of preparation 27.

M.p. ˜210° C.(dec.)

I.R. (Nujol): 3300, 2600-2400(shoulder), 1710 (shoulder), 1680, 1640cm⁻¹.

N.M.R. (DMSO-d₆), δ(ppm): 1.10-2.20(6H, m) 3.30 (1H, m), 3.65(2H, d, J=7Hz), 4.00(1H, m), 5.00(2H, s), 7.30(5H, s).

(2) Step 2 ##STR224##

Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOH (3) was prepared fromZ-(L)-mesoDAP-(L)-GlyOH (2) in substantially the same manner aspreparation 5-1.

N.M.R. (DMSO-d₆), δ(ppm): 1.30-2.30(15H, m), 3.80(2H, d, J=7 Hz),3.80-4.3(2H, m), 5.05(2H, s), 7.40(5H, s).

(3) Step 3 ##STR225##

Boc-(D)-mesoDAP-(L)-GlyOH (4) was prepared fromZ-(L)-Boc-(D)-mesoDAP-(L)-GlyOH (3) in substantially the same manner asthat of Preparation 20.

I.R. (Nujol): 3300, 2600-2400 (broad), 1680 (broad) cm⁻¹.

N.M.R. (D₂ O), δ(ppm): 1.10-2.20 (15H, m), 3.86 (2H, s), 3.80-4.20 (2H,m).

Preparation 55

(1) Step 1 ##STR226##

To a solution of Z-(L)-Boc-(D)mesoDAP-(D)-NHNHBoc (1)(5.72 g) andN-methylmorpholine (1.18 g) in methylene chloride (60 ml) was addedisobuthylchloroformate (1.60 g) at -10° to -15° C., and the mixture wasstirred at the same temperature for an hour. To the reaction mixture wasadded a solution of glycine trimethylsilyl ester (0.96 g) was added andstirred for 1.5 hours at -10° to -15° C. Methylene chloride was removedin vacuo, and the residue was dissolved in a mixture of ethyl acetate(60 ml) and 2.5% hydrochloric acid. The organic layer was washed with2.5% hydrochloric acid (50 ml) and water (50 ml) and dried overmagnesium sulfate. The solvent was removed in vacuo and the residue wastriturated successively with ethyl acetate (10 ml) and isopropylether(120 ml) and the precipitate was filtered and washed with isopropyletherto give Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (2)(6.1 g).

NMR (CDCl₃), δ(ppm): 1.43 (18H, s), 1.3-2.1 (6H, m), 3.9-4.5 (4H, m),5.10 (2H, s), 6.2-6.4 (4H, m), 7.10 (1H, broad s), 7.33 (5H, s), 8.95(1H, broad s).

(2) Step 2 ##STR227##

Boc(D)-mesoDAP(L)GlyOH-(O)-NHNHBoc (3) was obtained in substantially thesame manner as that of Preparation 20.

NMR (CD₃ OD), δ(ppm): 1.60 (9H, s), 1.63 (9H, s), 1.7-2.0 (6H, m), 3.92(2H, s), 3.8-4.1 (2H, m).

Preparation 56 ##STR228##

L-Lac(OAc)-L-Ala-D-Asp(OH)OBzl (3) was prepared from L-Ala-D-Asp(OH)OBzl(2) and acetyl-D-lactyl chloride (1) in substantially the same manner asthat of Preparation 22.

m.p. 109°-111° C. (dec.).

I.R. (Nujol): 3400, 3300, 1740, 1720, 1670 1550, 1520 cm⁻¹.

NMR (CDCl₃), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.45 (3H, d, J=7 Hz), 2.15(3H, s), 3.00 (2H, m), 4.4-5.4 (3H, m), 5.20 (2H, s), 7.16 (1H, d, J=8Hz), 7.33 (5H, s), 7.60 (2H, d, J=8 Hz), 8.56 (1H, s).

Preparation 57 ##STR229##

Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-Ala (1) (14.60 g) was dissolved in amixture of acetone (60 ml) and water (60 ml). The pH of the solution wasadjusted to about 8.0 with sodiumbicarbonate. The solution was stirredand cooled, and carbobenzyloxy chloride (5.30 g) was added thereto. Themixture was reacted for an hour at the same temperature. Removal ofacetone under reduced pressure gave an aqueous layer which was acidifiedwith 1N hydrochloric acid and extracted with ethyl acetate. The organiclayer was washed with water, dried over magnesium sulfate and evaporatedto give Z-(L)-Boc-(D)-MesoDAP-(D)-NHNHBoc-(L)-D-Ala (2) (14.60 g).

NMR (DMSO-d₆), δ(ppm): 1.0-2.0 (27H, m), 3.70-4.50 (3H, m), 5.03 (2H,s), 6.70 (1H, d, J=7 Hz), 7.20 (1H, broad), 7.33 (5H, s), 8.10 (1H, d,J=7 Hz), 8.63 (1H, s), 9.63 (1H, s).

Preparation 58 ##STR230##

A solution of mesoDAP N-carboxyanhydride (1) (17.0 g) in acetonitrile(130 ml) was cooled to 10° C. and a solution of cabobenzyloxyhydrazine(24.0 g) in acetic acid (80 ml) was added. The mixture was stirred for30 minutes at the same temperature. After concentration of the reactionmixture, the resulting residue was pulverized with ether to givemesoDAP-di-NHNHZ.2ACOH (2) (40.3 g).

m.p. 65°-70° C.

I.R. (Nujol): 3600-2200, 1700 cm⁻¹.

NMR (CD₃ OD) δ1.5-1.9 (6H, m), 1.92 (6H, s), 3.70 (2H, t, J=5 Hz), 5.13(4H, s), 7.38 (10H, s).

Acetic acid salt of mesoDAP-di-NHNHZ was dissolved in water andneutralized with 1N sodium hydroxide and then the resulting oil wascrystallized from water to give mesoDAP-di-NHNHZ.

m.p. 93°-95° C.

I.R. (Nujol): 3260, 1750, 1720, 1660 cm⁻¹.

NMR(CD₃ OD), δ1.5-1.8 (6H, m), 3.30 (2H, m), 5.13 (4H, s), 7.35 (10H,s).

Preparation 59 ##STR231##

A solution of mesoDAP-di-NHNHZ (1) (40.2 g) in a mixture of water (1.2l) and acetonitril (0.6 l) was adjusted to pH 8.5 with 1N sodiumhydroxide. To this solution was added leucine amino peptidase (from hogkidney) (Boehringer Manheim GmbH) (20 mg) and the mixture was shaken for6 hours at 37° C. After evaparation of methanol, the resulting aqueouslayer was washed with ethyl acetate and concentrated to about 200 ml.The concentrate was applied to a column of HP-20 resin (800 ml). Thecolumn was washed with water and eluted with 50% aqueous methanol. Theeluate was evaporated and the residue was pulverized with ether to givemesoDAP-(D)-NHNHZ (2) (15.0 g).

m.p. 205°-209° C. (dec).

I.R. (Nujol): 3600-2200, 1720, 1640, 1580 cm⁻¹.

NMR (CD₃ OD), δ1.4-2.1(6H, m), 3.50 (2H, m), 5.17 (2H, s), 7.40 (5H, s).

Preparation 60 ##STR232##

To a suspension of mesoDAP-(D)-NHNHZ (1.40 g) in methylene chloride (30ml) was added bis(trimethylsilyl)acetamide (6 ml) and the mixture wasstirred for 1 hour at room temperature. The resulting solution wascooled to -15° C. and carbobenzyloxy chloride (2.00 g) was added. Afterstirring for 1 hour at the same temperature, the mixture was poured intoa mixture of ethyl acetate (100 ml) and 2.5% hydrochloric acid (50 ml).The organic layer was separated, washed with saturated aqueous solutionof sodium chloride, dried over magnesium sulfate and evaporated. Theresidue was crystallized from chloroform to give di-Z-mesoDAP-(D)-NHNHZ(2) (2.20 g).

m.p. 139°-141° C.

I.R. (Nuj l): 3300, 1740, 1720, 1695, 1660 cm⁻¹.

NMR (CD₃ OD), δ: 1.40-2.0(6H, m), 4.10 (2H, m), 5.10 (6H, s), 7.33 (15H,s).

Preparation 61 ##STR233##

di-Z-mesoDAP-(D)-NHNHZ (1) (0.40 g) was added to thionyl chloride (4 ml)and the mixture was stirred for 30 minutes at room temperature.Evaporation gave Z-(D)-mesoDAP-(D)-NHNHZ-(L)-N-carboxyanhydride (2) asan oil.

I.R. (CH₂ Cl₂): 3400, 3270, 1850, 1780, 1700 cm⁻¹.

Preparation 62 ##STR234##

Z-(D)-mesoDAP-(D)-NHNHZ-(L)-N-carboxyanhydride (1), prepared from 0.40 gof di-Z-mesoDAP-(D)-NHNHZ as described above in Preparation 60, wasdissolved in a mixture of acetic acid (4 ml) and 1N hydrochloric acid (2ml) and the mixture was stirred for 6 hours at room temperature. Thereaction mixture was concentrated and the residue was dissolved in water(3 ml) and neutradized with dilute aqueous sodium bicarbonate. Theprecipitated crystalline solid was filtered and washed with water togive Z-(D)-mesoDAP(D)-NHNHZ (2) (0.25 g).

m.p. 205°-209° C. (dec).

I.R. (Nujol): 3300, 1715, 1690, 1665, 1605 cm⁻¹.

NMR (CF₃ COOH), δ: 1.6-2.5 (6H, m), 4.43 (2H, m), 5.20 (2H, s), 5.25(2H, s), 7.38 (10H, s).

Preparation 63 ##STR235##

A mixture of glycine (80 mg) and sodium carbonate (200 mg) in a mixtureof 1N sodium hydroxide (1 ml) and water (5 ml) was cooled to -5° C. anda solution of Z-(D)-mesoDAP-(D)-NHNHZ-(L)-N-carboxyanhydride, preparedfrom di-Z-mesoDAP-(D)-NHNHZ (1) (602 mg), in acetonitrile (5 ml) wasadded. The mixture was stirred for 2 hours at the same temperature andovernight at room temperature. The aqueous layer was separated and theorganic layer was extracted with 2% aqueous sodium chloride (5 ml). Theaqueous layers were combined and neutralized with 1N hydrochloric acid.The precepitated crystalline solid was filtered and washed with water togive Z-(D)-mesoDAP-(D)-NHNHZ-(L)-GlyOH (2) (120 mg).

m.p. 204°-7° C. (dec).

I.R. (Nujol): 3600-2200, 3250, 1720, 1700, 1690, 1660 cm⁻¹.

NMR (CF₃ CO₂ H) δ: 1.5-2.5 (6H, m), 4.0-4.6 (4H, m), 5.30 (4H, s), 7.38(10H, s).

Preparation 64 ##STR236##

A solution of phenylhydrazine (4.30 g) in acetic acid (10 ml) was cooledto 10° C. and a solution of mesoDAP N-carboxyanhydride (4.80 g) inacetonitrile (35 ml) was added. The mixture was stirred for 1 hour atthe same temperature and the solvent was evaporated. The residue waspulverized with ether to give mesoDAPdi-NHNHC₆ H₅ (acetic acid salt) (2)(10.10 g).

The compound (2) was dissolved in a mixture of water (300 ml) andmethanol (150 ml) and the pH was adjusted to 8.5. This solution wastreated with leucine amino peptidase (from Hog Kidney) (5 mg) at 37° C.for 5 hours. After evaporation of methanol, the resulting aqueous layerwas washed with ethyl acetate and concentrated to about 100 ml. Theconcentrate was applied to a column of HP-20 resin (200 ml). The columnwas eluted with 50% aqueous methanol. The eluate was evaporated to givemesoDAP-(D)-NHNHC₆ H₅ (3) (2.50 g).

I.R. (Nujol): 3600-2200, 1640, 1600 cm⁻¹.

NMR (CF₃ CO₂ H) δ: 1.9-2.6(6H), 4.0-4.5(2H, m), 7.43 (5H, s).

Preparation 65 ##STR237##

A mixture of mesoDAP-(D)-NHNHZ (1) (3.38 g) and cupric chloridedihydrate (853 mg) in 0.25N sodium hydroxide (50 ml) was cooled to 0° C.and di-t-butyldicarbonate (2.83 g) was added. The mixture was stirredfor 2 hours at the same temperature, during which time the pH of thereaction mixture was maintained at 9.5-10.0 by adding 1N sodiumhydroxide. Hydrogen sulfide was bubbled into the mixture for 10 minutesand the precipitate was filtered off. The filtrate was nentralized to pH5 with 1N hydrochloric acid and washed with ethyl acetate. The aqueouslayer was concentrated to about 10 ml and stood overnight at 5° C. Theprecipitated crystalline solid was filtered and washed with water togive Boc-(L)-mesoDAP-(D)-NHNHZ (2) (2.92 g).

m.p. 201°-3° C. (dec).

I.R. (Nujol): 3300, 3280, 1725, 1700 cm⁻¹.

NMR (CD₃ OD) δ: 1.40(9H,s), 1.3-2.1(6H, m), 3.8-4.1(2H,m), 5.17 (2H, s),7.38 (5H, s).

EXAMPLE 1

(1) Step 1 ##STR238##

L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(1) (662mg.) was dissolved in 50% aqueous acetone (8 ml.) and sodium bicarbonate(168 mg.) was added to the solution. To the mixture was added propionicchloride (408 mg.) at 0° C. and the resulting mixture was reacted at thesame temperature for an hour, maintaining the pH 7-8 with sodiumbicarbonate. The reaction mixture was adjusted to pH 3.0 with 1Nhydrochloric acid and concentrated to about 2 ml. in vacuo. To theresulting residue were added ethyl acetate (150 ml.), methanol (10 ml.)and brine (30 ml.). The organic layer was separated, dried over sodiumsulfate and then concentrated to dryness in vacuo. The concentrate wastreated with ether to givepropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(2) (400 mg) as a white powder.

IR (Nujol): 3250, 1720(shoulder), 1650 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 1.0-2.5 (36H, m), 3.97 (2H, s), 3.90-4.60 (4H, m).

Step (2) ##STR239##

Trifluoroacetic acid (1.8 ml) was added topropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(2) (350 mg) and the mixture was allowed to stand at ambient temperaturefor 15 minutes. The reaction mixture was evaporated in vacuo and theresidual oil was triturated with ether to give a white solid. The solidwas dissolved in water (10 ml) and to the solution were added 0.1Nsulfuric acid (12.2 ml) and a solution of sodium periodate (260 mg) inwater (4 ml) with stirring under ice-cooling. The stirring was continuedfor two hours and sodium bisulfite was added to the reaction mixtureuntil the iodine colour was disappeared. The resulting mixture wasevaporated to about 3 ml and the concentrate was adjusted to pH 2.5 with1N hydrochloric acid, and then chromatographed on a macroporousnon-ionic adsorption resin, HP20 (50 ml) (trade mark, maker: MitsubishiChemical Industry Co., Ltd.). Elution was carried out with a mixture ofwater and methanol (9:1). The fractions containing the object compound(3) were collected and freeze-dried to givepropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP-(L)-GlyOH (3) (102 mg) as awhite solid.

NMR (D₂)), δ(ppm): 1.11 (3H, t, J=8 Hz), 1.39 (3H, d, J=8 Hz), 2.31 (2H,q, J=8 Hz), 3.81 (1H, t, J=8 Hz), 3.96 (2H, s), 4.15-4.40 (3H, m)[α]_(D) =-37.0° (C=0.20 water).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Example 1, respectively.

EXAMPLE 2

(1) Step 1

Acetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D) mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3250, 1720, 1630, 1525 cm⁻¹.

NMR (CD₃ OH), δ(ppm): 2.00 (3H, s), 3.91 (2H, s).

(2) Step 2

Acetyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

mp 110°-114° C. (dec.).

[α]_(D) =-28.3° (C=0.36, water).

NMR (D₂ O), δ(ppm): 1.39 (3H, d, J=7 Hz), 2.03 (3H, s), 3.79 (1H, t, J=7Hz), 4.02 (2H, s), 4.20-4.50 (3H, m).

EXAMPLE 3

(1) Step 1

Heptanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3280, 1720, 1650, 1520 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 0.70-1.07 (3H, t), 3.92 (2H, s), 4.15-4.60 (3H,m).

(2) Step 2

Heptanoyl-L-Ala-γ-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

mp 175°-180° C.

NMR (D₂ O+NaHCO₃), δ(ppm): 0.83 (3H, t), 1.40 (3H, d, J=8 Hz), 3.77 (2H,s), 4.1-4.5 (3H, m).

EXAMPLE 4

(1) Step 1

Isobutyryl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3260, 1720, 1650 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 1.13 (3H, d, J=7 Hz), 1.24 (3H, d, J=7 Hz), 3.96(2H, s).

(2) Step 2

Isobutyryl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

NMR (D₂ O), δ(ppm): 1.07 (6H, d, J=7 Hz), 1.31 (3H, d, J=7 Hz), 2.50(1H, hept, J=7 Hz), 3.83 (1H, t, J=7 Hz), 3.98 (2H, s), 4.2-4.5 (3H, m).

[α]_(D) =-31.2° (C=0.17, water).

EXAMPLE 5

(1) Step 1

Benzoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol), δ(ppm): 3260, 1720, 1640 (broad), 1530 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 3.94 (2H, s), 7.45-7.70 (3H, m), 7.88-8.04 (2H,m).

(2) Step 2

Benzoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-4.2° (C=0.19, water).

NMR (D₂ O), δ(ppm): 1.53 (3H, d, J=7 Hz), 3.83 (1H, t, J=7 Hz), 3.98(2H, s), 4.15-4.60 (3H, m), 7.40-7.65 (3H, m), 7.70-7.90 (2H, m).

EXAMPLE 6

(1) Step 1

D-mandelyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3280, 1720, 1650, 1520 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 3.94 (2H, s), 4.25-4.55 (3H, m), 5.12 (1H, s),7.40 (5H, m).

(2) Step 2

D-mandelyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-40.5 (C=0.19, water).

NMR (D₂ O), δ(ppm): 1.46 (3H, d, J=7 Hz), 3.83 (1H, t, J=7 Hz), 3.97(2H, s), 4.13-4.50 (3H, m), 5.23 (1H, s), 7.48 (5H, s).

EXAMPLE 7

(1) Step 1

2-Methoxypropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

NMR (CD₃ OD), δ(ppm): 3.41 (3H, s), 3.80 (1H, q, J=7 Hz), 3.95 (2H, s),4.25-4.55 (3H, m).

(2) Step 2

2-Methoxypropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-30.0° (C=0.21, water).

NMR (D₂ O), δ(ppm): 1.33 (3H, d, J=7 Hz), 1.41 (3H, d, J=7 Hz), 3.36(3H, s), 3.75-4.03 (2H, m), 3.96 (2H, s), 4.2-4.5 (3H, m).

EXAMPLE 8

(1) Step 1

Phenoxyacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3280, 1650, 1520 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 3.92 (2H, s), 4.58 (2H, s), 6.85-7.10 (3H, m),7.20-7.40 (2H, m).

(2) Step 2

Phenoxyacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-19.6° (C=0.23, water).

NMR (D₂ O), δ(ppm): 1.43 (3H, d, J=7 Hz), 3.82 (1H, t, J=7 Hz), 3.96(2H, s), 4.20-4.55 (3H, m), 4.64 (2H, s), 6.97-7.15 (3H, m), 7.30-7.43(2H, m).

EXAMPLE 9

(1) Step 1 ##STR240##

Acetyloxyacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(2)(570 mg) was prepared fromL-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH (1) (794mg) and acetylglycolic chloride of (246 mg) in substantially the samemanner as that Step 1 of Example 1.

I.R (Nujol): 3270, 1710, 1650, 1525 cm⁻¹.

NMR (DMDO-d₆), δ(ppm): 1.24 (3H, d, J=7 Hz), 2.08 (3H, s).

(2) Step 2 ##STR241##

Acetyloxyacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(2) (490 mg) was dissolved in 50% aqueous methanol (20 ml) and thesolution was adjusted to pH 9.0 with 5% potassium carbonate and thenallowed to stand at ambient temperature for an hour. The solution wasadjusted to pH 5 and concentrated in vacuo and then water (1 ml) wasadded to the concentrate. The mixture was adjusted to pH 2.5 with 1Nhydrochloric acid and chromatographed on a column of macroporousnon-ionic adsorption resin, HP 20 (20 ml). After washing the column withwater, elution was carried out with 50% aqueous methanol. The fractionscontaining the object compound (3) were collected and concentrated togivehydroxyacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(3) (370 mg) as a white solid.

I.R. (Nujol): 3300, 1720 (shoulder), 1650, 1530 cm⁻¹.

NMR (D₂ O), δ (ppm): 3.95 (2H, s), 4.112H, s).

(3) Step 3 ##STR242##

Hydroxyacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP-(L)-GlyOH (4) (234 mg)was prepared from hydroxyacetyl-L-AAla-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH (3) (345mg) in substantially the same manner as that of Step 2 of Example 1.

[α]_(D) =-25.2° (C=0.21, water).

NMR (D₂ O), δ (ppm): 1.43 (3H, d, J=7 Hz), 3.91 (1H, t, J=7 Hz), 3.99(2H, s), 4.15 (2H, s), 4.20-4.55 (3H, m).

EXAMPLE 10

The following compounds were prepared in substantially the same manneras that of steps 1 and 2 of Example 1.

(1) Step 1

L-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP(L)-GlyOH-(D)-NHNHBoc.

I.R(Nujol): 3300, 1720, 1650 cm⁻¹.

NMR (CD₃ OD), δ (ppm): 2.13 (3H, s), 3.93 l (2H, s).

(2) Step 2

L-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

mp 170°-174° C. (dec.).

[α]_(D) =-33.2° (C=0.25, water).

NMR (D₂ O), δ (ppm): 1.38 (3H, d, J=7 Hz), 1.45 (3H, d, J=7 Hz), 3.89(1H, t, J=7 Hz), 4.01 (2H, s), 4.20-4.55 (4H, m).

EXAMPLE 11

(1) Step 1 ##STR243##

To a mixture of Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1) (0.922 g) andtriethylamine (0.202 g) in 50% aqueous dioxane (30 ml) was addedBoc-D-Ala-L-Ala-D-Glu(oSu)oBzl. The resulting mixture was stirred atambient temperature overnight and then evaporated. The residue wasadjusted to pH 8 with dil sodium bicarbonate and washed with ethylacetate. The aqueous layer was acidified to pH 1 with dil hydrochloricacid and then extracted with ethyl acetate. The extract was washed withwater, dried over magnesium sulfate and evaporated. The residue wascrystallized from a mixture of ethyl acetate and ether, and thenfiltered. The crystal was washed with ether to giveBoc-D-Ala-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.45 g).

NMR (CD₃ OD), δ (ppm): 1.29 (3H, d, J=7 Hz), 1.32 (3H, d, J=7 Hz), 1.40(27H, s), 3.87-4.47 (4H, m), 5.14 (2H, s), 7.32 (5H, s).

(2) Step 2 ##STR244##

A solution of Boc-D-Ala-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH (3) (1.2 g) in a mixture (25 ml) of methanoland water (2:1) was hydrogenated over 5% palladium-black (0.2 g) at twoatmospheric pressures of hydrogen gas. After the reaction was completed,the catalyst was filtered off and the filtrate was evaporated. Theresidue was pulverized with isopropyl ether and filtered to giveBoc-D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(4) (1.1 g).

NMR (CD₃ OD), δ (ppm): 1.24 (3H, d, J=7 Hz), 1.35 (3H, d, J=7 Hz), 1.44(27H, s), 1.34-2.50 (10H, m), 3.94 (2H, s), 3.87-4.50 (4H, m).

(3) Step 3 ##STR245##

Boc-D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(4) (1.0 g) was dissolved in trifluoroacetic acid (5 ml) and thesolution was stirred at ambient temperature for 30 minutes. The reactionmixture was evaporated and the residue was pulverized with ethyl acetateand filtered to give D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP-(D)-NHNH₂-(L)-GlyOH (5) trifluoroacetic acid salt (0.81 g).

NMR (D₂ O), δ (ppm): 1.42 (3H, d, J=7 Hz), 1.57 (3H, d, J=7 Hz),1.34-2.50 (10H, m), 3.74 (2H, s), 3.67-4.27 (4H, m).

(4) Step 4 ##STR246##

N-Bromosiccinimide (178 mg) was added to a solution ofD-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP-(D)-NHNH₂ -(L)-GlyOH (5)trifluoroacetic acid salt (532 mg) in 50% aqueous dioxane (10 ml). Themixture was stirred at ambient temperature for an hour and adjusted topH 3.5 with dil sodium bicarbonate and then evaporated. The residue wasdissolved in water and chromatographed on a column of a macroporousnon-ionic adsorption resin, HP 20 (150 ml) and then eluted with water.Eluates containing the object compound (6) were combined and evaporated.The residue was washed with methanol and dissolved in water. Thesolution was adjusted to pH 3.0 and chromatographed again on a column ofmacroporous non-ionic adsorption resin, HP 20 (100 ml) and eluted withwater. Fractions containing the object compound (6) were combined andevaporated to give a solid. The solid was dissolved in water andlyophilized to give D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP-(L)-GlyOH (6)(120 mg).

NMR (D₂ O), δ (ppm): 1.45 (3H, d, J=7 Hz), 1.57 (3H, d, J=7 Hz),1.34-2.34 (10H, m), 3.88 (2H, s), 3.74-4.50 (4H, m).

EXAMPLE 12

(1) Step 1 ##STR247##

D-Lac-(oAc)-Gly-γ-D-GluoBzl dicyclohexylamine salt (2) (1.20 g) wasdissolved in a mixture of ethyl acetate (40 ml) and methylene chloride(10 ml). To the solution was added 1N hydrochloric acid (2 ml) and theprecipitates thus formed were filtered off. The organic layer was washedwith water, dried over magnesium sulfate and then evaporated to give anoily residue. The residue was dissolved in methylene chloride (20 ml)and to the solution were added N-methyl morpholine (200 mg) and isobutylchlorocarbonate (270 mg) at 0° C. The mixture was reacted at the sametemperature for 20 minutes. To the reaction mixture was added a solutionof Boc-(D)-mesoDap-(D)-NHNHBoc-(L)-GlyOH (1) (860 mg) in a mixture ofmethylene chloride (40 ml) and dimethylformamide (50 ml) containingbis(trimethylsilyl)acetamide (2 ml) at 0° C. The resulting mixture wasstirred at the same temperature for two hours and concentrated in vacuoto give an oily residue. The residue was dissolved in ethyl acetate andthe solution was washed with 2% hydrochloric acid. The organic layer waswashed with successively with water and aqueous saturated sodiumchloride solution and dried and then evaporated. The residue wasthroughly washed with ether and collected by filtration to giveD-Lac(oAc)-Gly-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-Gly(OH)(3) (1.0 g).

NMR (DMSO-d₆), δ (ppm): 0.80-2.60 (3H, m), 2.06 (3H, s), 3.76 (4H, broads), 3.60-4.52 (3H, m), 5.00 (1H, q, J=7 Hz), 5.12 (2H, s), 6.70 (1H, d,J=7 Hz), 7.38 (5H, s), 7.80-8.80 (5H, m), 9.58 (1H, s).

(2) Step 2 ##STR248##

D-Lac(oAc)-Gly-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (0.9 g) was dissolved in glacial acetic acid (9 ml) and hydrogenatedover 10% palladium black (200 mg) at ambient temperature for 1.5 hours.The catalyst was filtered off and the filtrate was evaporated in vacuoto give an oily residue which was pulverized with ether to giveD-Lac(oAc)-Gly-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4) (0.76 g).

NMR (DMSO-d₆), δ (ppm): 0.80-2.80 (31H, m), 2.08 (3H, s), 3.72 (4H,broad s), 3.60-4.36 (3H, m), 5.00 (1H, q, J=7 Hz), 6.76 (1H, m),7.72-8.40 (5H, m), 8.70 (1H, m), 9.60 (1H, s).

(5) Step 3 ##STR249##

D-Lac(oAc)-Gly-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4) (0.70 g) was dissolved in 50% aqueous methanol (20 ml) and thesolution was adjusted to pH 9.0 with 5% aqueous potassium carbonate. Thesolution was allowed to stand at ambient temperature for 3 hours andadjusted to pH 7.0 with 5% hydrochloric acid. The solution waschromotographed on a column of macroporous non-ionic adsorption resin,HP 20 (25 ml) and eluted successively with water (100 ml) and 50%aqueous methanol (100 ml). The latter fraction was evaporated to give aposty residue which was pulverized with ether to giveD-Lac-Gly-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (5)(0.40 g),

I.R. (Nujol): 3300, 1730, 1690 1650 (broad) cm⁻¹.

NMR (D₂ O), δ (ppm): 1.0-2.60 (31H, m), 4.00 (4H, s), 4.20-4.60 (4H, m).

(4) Step 4 ##STR250##

D-Lac-Gly-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (5)(335 mg) was dissolved in trifluoroacetic acid (3 ml) and the solutionwas stirred at ambient temperature for 15 minutes. The solution wasevaporated in vacuo and the residue thus obtained was pulverized withether to give a white powder. The powder was dissolved in a mixture ofwater (8 ml) and 0.1N sulfuric acid (11.6 ml) and the solution wascooled in an ice-bath. To the solution was added an aqueous sodiummetaperiodate solution (240 mg in 3 ml of water) and allowed to react at0° C. for an hour. The resulting reaction mixture was treated with anaqueous sodium bicarbonate until the purple color of the solution wasdisappeared. The solution was adjusted to pH 3.0 and concentrated toabout 3 ml. The concentrate was adjusted to pH 2.0 and thenchromatographed on a column of macroporous non-ionic adsorption resin,HP 20 (80 ml) and eluted with water. Fractions containing the objectcompound (6) were collected and evaporated in vacuo to give a postyresidue which was dissolved in water and lyophilized to giveD-Lac-Gly-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (6) (0.150 g).

[α]_(D) =-5.0° (C=0.2 in water)

NMR (D₂ O), δ (ppm): 1.38 (3H, d, J=7 Hz), 1.20-2.60 (10H, m), 3.82 (1H,t, J=7 Hz), 3.96 (2H, s), 4.00 (2H, s), 3.68-4.52 (3H, m).

EXAMPLE 13

(1) Step 1 ##STR251##

D-Lac(oAc)-L-Ser(oBzl)-D-GluoBzl (2) dicyclohexylamine salt (1.40 g) wasdissolved in a mixture of ethyl acetate (40 ml) and chloroform (10 ml).To the solution was added 1H hydrochloric acid (2 ml) and theprecipitates thus formed were filtered off. The organic layer wasseparated, washed with water, dried over magnesium sulfate and thenevaporated to give an oily residue (1.08 g). The residue was dissolvedin methylene chloride (20 ml) and the solution was treated withN-methylmorpholine (198 mg) and cooled to -10° C. and thenisobutylchlorocarbonate (270 mg) was added thereto. To the mixture wasadded Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1) (860 mg) in a mixture ofmethylene chloride (40 ml) and dimethylformamide (50 ml) containingbis(trimethylsilyl)acetamide (2 ml). The resulting mixture was allowedto react at -10° C. for two hours. The reaction mixture was concentratedin vacuo to give an oily residue which was dissolved in ethyl acetate.The solution was washed with 2% aqueous hydrochloric acid and the ethylacetate layer was separated, washed with water and dried over magnesiumsulfate, and then evaporated. The crystalline residue thus obtained waswashed with ether to giveD-Lac-(oAc)-L-Ser(oBzl)-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.55 g).

NMR (DMSO-d₆), δ (ppm): 1.00-2.36 (10H, m), 1.38 (18H, s), 1.32 (3H, d,J=7 Hz), 2.04 (3H, s), 3.44-4.84 (8H, m), 4.48 (2H, s), 5.06 (1H, q, J=7Hz). 5.14 (2H, s), 6.72 (1H, broad s), 7.34 (5H, s), 7.40 (5H, s), 7.92(1H, broad s), 8.04-8.44 (4H, m), 8.72 (1H, broad s), 9.60 (1H, s).

Successively, the following compounds were prepared in substantially thesame manner as that of Steps 2 to 4 of Example 12, respectively.

(2) Step 2

D-Lac(oAc)-L-Ser(OH)-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH

NMR (DMSO-d₆), δ (ppm): 1.00-2.40 (10H, m), 1.38 (18H, s), 1.32 (3H, d,J=7 Hz), 2.08 (3H, s), 3.40-4.52 (8H, m), 5.06 (1H, q, J=7 Hz), 6.76(1H, d, J=7 Hz), 7.64-8.32 (4H, m), 8.72 (1H, s), 9.60 (1H, s).

(3) Step 3

D-Lac-L-Ser-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

NMR (D₂ O), δ (ppm): 1.00-2.60 (10H, m), 1.42 (3H, d, J=7 Hz), 1.44(18H, s), 3.60-5.00 (9H, m).

(4) Step 4

D-Lac-L-Ser-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-18.5° (C=0.2 water)

NMR (D₂ O), δ (ppm): 1.20-2.60 (10H, m), 1.40 (3H, d, J=7 Hz), 3.60-5.10(7H, m), 3.98 (2H, s).

EXAMPLE 14

(1) Step 1 ##STR252##

D-Lac-(oAc)-L-Val-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.20 g) was prepared from Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1)(800 mg) and D-Lac(oAc)-L-Val-γ-D-GluoBzl (2) (0.90 g) in substantiallythe same manner as that of Step 1 of Example 12.

NMR (DMSO-d₆), δ (ppm): 0.80 (3H, d, J=7 Hz), 0.84 (3H, d, J=7 Hz), 1.83(18H, s), 1.0-2.40 (14H, m), 2.04 (3H, s), 3.60-4.48 (6H, m), 5.08 (1H,q, J=7 Hz), 5.14 (5H, s), 6.74 61H, d, J=7 Hz), 7.36 (5H, s), 7.80-8.40(5H, m), 8.68 (1H, s), 9.54 (1H, s).

Successively, the following compounds were prepared in substantially thesame manner as that of Steps 2 to 4 of Example 12, respectively.

(2) Step 2

D-Lac-(oAc)-L-Val-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

NMR (DMSO-d₆), δ (ppm): 0.82 (3H, d, J=7 Hz), 0.86 (3H, d, J=7 Hz),1.00-2.40 (14H, m), 2.04 (3H, s), 3.60-4.40 (6H, m), 5.04 (1H, q, J=7Hz), 6.76 (1H, d, J=7 Hz), 7.80-8.40 (4H, m), 8.70 (1H, s), 9.60 (1H,s).

(3) Step 3

D-Lac-L-Val-γ-D-GLu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

NMR (D₂ O), δ (ppm); 0.96 (6H, d, J=7 Hz), 1.12-2.56 (32H, m), 3.96 (2H,s), 3.80-4.52 (5H, m).

(4) Step 4

D-Lac-L-Val-γ-D-Glu(α-OH)-(L)-mesoDAP(L)-GlyOH.

[α]_(D) =-28.0° (C=0.2, water).

NMR (D₂ O), δ (ppm): 0.96 (6H, d, J=7 Hz), 1.37 (3H, d, J=7 Hz),1.20-2.60 (11H, m), 3.96 (2H, s), 3.68-4.50 (5H, m).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Example 1, respectively.

EXAMPLE 15

(1) Step 1

Thienylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

NMR (CD₃ OD), δ (ppm): 3.76 (2H, s), 3.87 (2H, s), 4.15-4.40 (3H, m),6.90 (2H, m), 7.20 (1H, m).

(2) Step 2

Thienylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH

[α]_(D) =-33.5° (C=0.20 water)

NMR (D₂ O), δ (ppm): 1.40 (3H, d, J=7 Hz), 3.81 (3H, t, J=7 Hz), 3.89(2H, s), 3.97 (2H, s), 4.13-4.44 (3H, m), 7.02 (2H, m), 7.35 (1H, m).

EXAMPLE 16

(1) Step 1

Phenylcarbamoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3300, 1730, 1650, 1595, 1540 cm⁻¹.

NMR (CD₃ OD), δ (ppm): 3.95 (2H, s), 4.2-4.6 (3H, m), 6.9-7.1 (5H, m).

(2) Step 2

Phenylcarbamoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-38.5° (C=0.20, DMSO).

NMR (D₂ O+NaHCO₃), δ (ppm): 1.43 (3H, d, J=7 Hz), 3.96 (1H, m), 3.76(2H, s), 4.05-4.5 (3H, m), 7.35 (5H, m).

EXAMPLE 17

(1) Step 1 ##STR253##

Triethylamine (720 mg) and benzyloxycarbonyl-L-Ala-D-Glu(oSu)oBzl (1)(3.51 g) was added to a solution ofBoc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH (2) (3.00 g) in a mixture ofdioxane (90 ml) and water (70 ml). The resulting mixture was allowed tostand at ambient temperature for 18 hours, and concentrated to about 50ml. The concentrate was adjusted to pH 3 with 1N hydrochloric acid andextracted with ethyl acetate (300 ml) containing methanol (20 ml). Theorganic layer was washed with brine (100 ml), dried over sodium sulfateand then evaporated in vacuo. The residue was washed with ether andfiltered to givebenzyloxycarbonyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(3) (5.53 g).

NMR (DMSO-d₆), δ (ppm): 1.07 (3H, d, J=7 Hz), 3.64-4.48 (6H, m), 5.04(2H, s), 5.12 (2H, s), 6.56 (1H, d, J=8 Hz), 7.35 (10H, s), 7.95 (1H, d,J=8 Hz), 8.15 (1H, m), 8.35 (1H, d, J=8 Hz), 8.70 (1H, s), 9.56 (1H, s).

(2) Step 2 ##STR254##

Benzyloxycarbonyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.43 g) was added to trifluoroacetic acid (6 ml) and the mixturewas stood at ambient temperature for an hour. The mixture was evaporatedin vacuo and the residual oil was triturated with isopropyl ether togive a solid. The solid was dissolved in a mixture of water (35 ml) andmethanol (10 ml). To this solution were added 1N sulfuric acid (40 ml)and sodium periodate (860 mg) at 0° C. The mixture was stirred at thesame temperature for 1.5 hours and concentrated to 10 ml and adjusted topH 4 with 1N sodium hydroxide. The precipitate thus obtained wascollected and dried over phosphorus pentoxide to givebenzyloxycarbonyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(L)-GlyOH (4) (109g).

I.R. (Nujol): 3300, 1700, 1660, 1540 cm⁻¹.

NMR (D₂ O+NaHCO₃), δ(ppm): 3.78 (2H, s), 5.04 (2H, s), 5.13 (2H, s),7.40 (10H, m).

(3) Step 3 ##STR255##

To a suspension ofbenzyloxycarbonyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(L)-GlyOH (4) (600mg) in water (10 ml) was added 1N sodium hydroxide (35 ml). The mixturewas stood at ambient temperature for an hour. The reaction mixture wasadjusted to pH 2.0 with 1N hydrochloric acid and concentrated to about 3ml. The residual solution was chromatographed on a column of amacroporous non-ionic adsorption resin, HP 20 (60 ml) and eluted with ina mixture of water and methanol (7:3). The fraction containing theobject compound (5) was collected and concentrated to dryness in vacuoto give benzyloxycarbonyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (5)(190 mg).

mp 205°-207° C.

[α]_(D) =-12.1° (C=0.19, water).

NMR (D₂ O), δ(ppm): 1.37 (3H, d, J=7 Hz), 3.78 (1H,m), 3.93 (2H, s),4.05-4.40 (3H, m), 5.14 (2H, s), 7.45 (5H, s).

EXAMPLE 18

(1) Step 1 ##STR256##

To a mixture of1-O-α-benzyl-4,6-O-benzylidene-N-acetyl-muramyl-L-Ala-γ-D-GluoBzl (2)(0.76 g) and triethylamine (0.10 g) in methylene chloride (5 ml) wasadded dropwise iso-butyl chloroformate (0.13 g) at -18° C. The resultingmixture was stirred at -15° C. for 50 minutes and to this mixture wasadded at -28° C. silyl ester of Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(1) which was prepared from Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GLyOH (0.46g) and bis(trimethylsilyl)acetamide in a mixture (5 ml) of methylenechloride and dimethylformamide (5:1) with stirring at ambienttemperature. The mixture was stirred at -15° C. for 1.5 hours andallowed to warm up to ambient temperature. The solution was distilledoff and to the residue thus obtained was added 2% aqueous hydrochloricacid (10 ml) to give a powder. The powder was collected by filtrationand washed with water to give1-O-α-benzyl-4,6-O-benzylidene-N-acetyl-muramyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.14 g).

NMR (DMSO-d₆), δ(ppm): 1.48 (18H, s), 1.1-1.6 (6H, m), 1.85 (3H, s),1.8-2.3 (8H, m), 3.7-4.0 (8H, m), 4.0-4.5 (5H, m), 4.7 (2H, m), 4.90(1H, d, J=7 Hz), 5.15 (2H, s), 5.72 (1H, s), 7.33 (5H, s), 7.40 (5H, s),7.34 (5H, s), 8.1-8.3 (4H, m), 8.3-8.7 (3H, m).

(2) Step 2 ##STR257##

1-O-α-benzyl-4,6-O-benzylidene-N-acetylmuramyl-L-Ala-γ-D-Glu(.alpha.-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (0.81 g) was added to trifluoroacetic acid (4 ml) and the mixturewas stirred at ambient temperature for 15 minutes. The solvent wasdistilled off and the residue was pulvelized with ether and filtered togive1-O-α-benzyl-N-acetyl-muramyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(D)-NHNH₂-(L)-GlyOH (4) trifluoroacetic acid salt (0.65 g).

NMR (D₂ O), δ(ppm): 1.38 (6H, d, J=7 Hz), 1.84 (3H, s), 1.2-1.5 (2H, m),1.7-2.1 (6H, m), 2.2-2.4 (2H, m) 3.6-4.0 (8H, m), 4.2-4.5 (5H, m), 5.18(2H, s), 7.4 (10H, s).

(3) Step 3 ##STR258##

To a solution of1-O-α-benzyl-N-acetylmuramyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(D)-NHNH₂-(L)-GlyOH (4) trifluoroacetic acid salt (0.58 g) in 60% aqueous aceticacid was added manganese dioxide (0.15 g) and the mixture was stirred atambient temperature for 4 hours. The reaction mixture was filtered andthe filtrate was concentrated. The concentrate was dissolved in water (3ml) and the solution was adjusted to pH 8 with conc. ammonium hydroxide.To the solution was added methanol (3 ml) and the mixture was allowed tostand in a refrigerator. An additional 14 ml of 5% aqueous methanol wasadded to the mixture and the resulting mixture was filtered.

The filtrate was concentrated and the concentrate was dissolved in 1Nacetic acid (30 ml) and the solution was put on a column of a chelateresin. The column was eluted with water and the fractions containing theobject compound (5) were collected and concentrated to give an oil. Theoil was dissolved in water and the solution was adjusted to pH 2.6 andput on a column of a macroporous non-ionic adsorption resin, HP 20. Thecolumn was washed with water and eluted with 50% aqueous methanol. Thefractions containing the object compound (5) were combined andconcentrated to give1-O-α-benzyl-N-acetylmuramyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(L)-GlyOH(5)(0.25 g).

NMR (D₂ O), δ(ppm): 1.35 (3H, d, J=7 Hz), 1.38 (3H, d, J=7 Hz), 1.2-1.6(2H, m), 1.80 (3H, s), 1.6-2.0 (6H, m), 2.2-2.4 (2H, m), 3.5-3.9 (8H,m). 4.2-4.5 (5H, m), 4.90 (1H, d, J=7 Hz), 5.20 (2H, s), 7.38 (5H, s),7.40 (5H, s).

(4) Step 4 ##STR259##

A solution of1-O-α-benzyl-N-acetylmuramyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(L)-GlyOH(5) (0.20 g) in a mixture of water (20 ml) and acetic acid (1.5 ml) washydrogenated over 10% palladium black (0.10 g) under an atmosphericpressure of hydrogen. The catalyst was filtered off and the filtrate wasevaporated to give a white solid (0.15 g). The solid was dissolved inwater (2 ml) and lyophilized to giveN-acetylmuramyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (6) (0.12 g).

NMR (D₂ O), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.41 (3H, d, J=7 Hz), 1.2-1.6(2H, m), 1.99 (3H, s), 1.6-2.2 (6H, m), 2.2-2.4 (2H, m), 3.4-4.0 (5H,m), 3.82 (2H, s), 4.1-4.5 (6H, m), 5.19 (1H, m).

EXAMPLE 19

(1) Step 1 ##STR260##

D-Lac(oAc)-L-Phe-D-GluoBzl (2) (1.0 g) was dissolved in methylenechloride (40 ml) and N-methylmorpholine (200 mg) was added thereto. Tothis stirred solution cooled at -10° C. in an ice salt-bath,isobutylchlorocarbonate (270 mg) was added and the mixture was allowedto react at the same temperature for 20 minutes. To the reaction mixturewas added the silyl ester prepared fromBoc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1) (920 mg) in a mixture ofmethylene chloride (20 ml) and dimethylformamide (10 ml) containingbis(trimethylsilyl)acetamide (2 ml). The reaction mixture was stirredfor 2 hours and concentrated under reduced pressure to give an oilyresidue which was dissolved in ethyl acetate and washed with 2%hydrochloric acid. The organic layer was washed successively with waterand saturated sodium chloride and then dried. Evaporation of the solventgave a residue which was thoroughly washed with ether and collected byfiltration to giveD-Lac(oAc)-L-Phe-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.660 g).

NMR (DMSO-d₆), δ(ppm): 1.10 (2H, d, J=7 Hz), 1.23-2.43 (28H, m), 2.00(3H, s), 2.86-3.23 (2H, m), 3.73-4.70 (6H, m), 5.17 (2H, s), 7.23 (5H,s), 7.36 (5H, s), 7.70-8.90 (5H, m) 9.60 (1H, s).

(2) Step 2 ##STR261##

D-Lac(oAc)-L-Phe-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (1.50 g) was dissolved in 50% aqueous methanol (20 ml). To thissolution was added 1N sodium hydroxide (5 ml) and the resulting solutionwas allowed to stand at ambient temperature for 2 hours. The solutionwas concentrated to about 3.0 ml after adjusting the pH of the solutionto pH 7.0. The pH of the solution was adjusted again to about 2.0 andthe precipitates thus formed was extracted with ethyl acetate. Theorganic layer was washed with saturated sodium chloride and dried overmagnesium sulfate and then the solvent was evaporated to giveD-Lac(OH)-L-Phe-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4) (0.960 g).

NMR (DMSO-d₆), δ(ppm): 1.12 (3H, d, J=7 Hz), 1.0-2.32 (28H, m),2.72-3.20 (2H, m), 3.60-4.80 (7H, m), 6.72 (1H, m), 7.20 (5H, s),7.48-8.80 (5H, m), 9.52 (1H, s).

(3) Step 3 ##STR262##

D-Lac(OH)-L-Phe-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4) was dissolved in trifluoroacetic acid (5 ml) and the solution wasstirred at ambient temperature for 15 minutes. Evaporation of thesolvent under reduced pressure gave an oily residue, which waspulverized with ether to give a white powder. The powder was dissolvedin a mixture of water (15 ml) and 0.1N sulfuric acid and the solutionwas cooled in an ice-bath. To this stirred solution was added an aqueoussodium periodate solution (528 mg in 8 ml of water) and the mixture wasallowed to react at the same temperature for an hour. The resultingreaction mixture was treated with sodium bisulfite until the brown colorof the mixture became clear and the pH of the solution was adjusted toabout 3.0. Then, the solution was concentrated to about 4 ml and the pHof the solution was adjusted again to pH 2.0. The solution was appliedto a column of a macroporous non-ionic adsorption resin, HP 20 (60 ml)and eluted with water. Evaporation of fractions containing the objectcompound (5) was carried out to giveD-Lac(OH)-L-Phe-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (5) (0.45 g).

[α]_(D) =-5.0° (C=0.2, water).

NMR (D₂ O), δ(ppm): 1.28 (3H, d, J=7 Hz), 1.32-2.28 (10H, m), 3.08 (2H,ABq, J=16 Hz), 3.80 (1H, t, J=7 Hz), 3.96 (2H, s), 4.0-4.44 (4H, m),7.32 (5H, s).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Example 1, respectively.

EXAMPLE 20

(1) Step 1

Pivaloyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.22 (9H, s), 1.38 (2H, d, J=7 Hz), 1.48 (18H,s), 1.34-2.42 (10H, m), 3.95 (2H, s), 4.27-4.60 (4H, m).

(2) Step 2

Pivaloyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

N.M.R. (D₂ O), δ(ppm): 1.20 (9H, s), 1.37 (3H, d, J=7 Hz), 1.34-2.42(10H, m), 3.80 (2H, s), 4.10-4.45 (4H, m).

EXAMPLE 21

(1) Step 1

Adamantane-1-carbonyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3280, 1720 (shoulder), 1630, 1520 cm⁻¹.

(2) Step 2

Adamantane-1-carbonyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH

[α]_(D) =-18.9° (c=0.18, water).

N.M.R. (D₂ O), δ(ppm): 1.46 (3H, d, J=7 Hz), 3.83 (1H, t, J=7 Hz), 3.98(2H, s), 4.25-4.5 (3H, m).

EXAMPLE 22

(1) Step 1

Lauroyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GLyOH.

I.R. (Nujol): 3300, 1730 (shoulder), 1650, 1520 cm⁻¹.

N.M.R. (CD₃ OD), δ(ppm): 0.96 (3H, m), 3.96 (2H, s).

(2) Step 2

Lauroyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-30.0° (c=0.14, 1N NaOH).

N.M.R. (D₂ O+NaHCO₃), δ(ppm): 0.88 (3H, m), 3.65-3.85 (3H, m), 4.10-4.60(3H, m).

EXAMPLE 23

(1) Step 1

Salicyloyl(oBzl)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.42 (9H, s), 1.44 (9H, s), 3.87 (2H, s), 5.23(2H, s), 6.93-7.55 (8H, m), 7.94 (1H, d,d, J=2 and 7 Hz).

(2) Step 2

Salicyloyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH

[α]_(D) =4.6° (c=0.13 water).

N.M.R. (D₂ O), δ(ppm): 1.51 (3H, d, J=7 Hz), 3.77 (1H, t, J=7 Hz), 3.91(2H, s), 6.97-7.10 (2H, m), 7.40-7.55 (1H, m), 7.71-7.79 (1H, m).

EXAMPLE 24

(1) Step 1

α-ethylhexanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm), δ(ppm): 0.88 (6H, t, J=7 Hz), 1.42 (9H, s),1.54 (9H, s), 1.80-2.36 (19H, m), 3.94 (2H, s).

(2) Step 2

α-ethylhexanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-46.7 (c=0.12, water).

N.M.R. (D₂ O), δ(ppm): 0.81 (6H, t, J=7 Hz), 1.37 (2H, d, J=7 Hz), 3.81(1H, t, J=7 Hz), 3.96 (2H, s), 4.2-4.5 (3H, m).

EXAMPLE 25

(1) Step 1

Nicotinoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.40 (18H, m), 3.90 (2H, s), 7.50 (1H, d,d, J=8and 5 Hz), 8.31 (1H, m), 8.69 (1H, m), 9.11 (1H, m).

(2) Step 2

Nicotinoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-7.7° (c=0.22, water).

N.M.R. (D₂ O), δ(ppm): 1.56 (3H, d, J=7 Hz), 3.83 (1H, t, J=6 Hz), 3.95(2H, s), 7.94 (1H, d,d, J=8 and 4 Hz), 8.85 (1H, m), 9.10 (1H, m),

EXAMPLE 26

(1) Step 1

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH.

I.R. (Nujol): 3300, 1720, 1630, 1530 cm⁻¹.

N.M.R. (CD₃ OD+CDCl₃), δ(ppm): 0.80 (3H, m) 3.95 (2H, s).

(2) Step 2

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-30.8° (c=0.13, 1N NaOH).

I.R. (Nujol): 3280, 1730, 1630, 1530 cm⁻¹, NMR.

(NaoD--D₂ O) δ(ppm) 0.83 (3H, m), 3.24 (1H, m), 3.82 (2H, s).

EXAMPLE 27

(1) Step 1 ##STR263##

To a mixture of Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1) (0.46 g) andtriethylamine (0.11 g) in a mixture (20 ml) of dioxane and water (2:1)was added benzoyl-D-Ala-L-Ala-D-Glu(γ-OSu)oBzl (2) (0.58 g).

The resulting mixture was stirred at ambient temperature for 18 hours.After evaporation of dioxane, the remaining aqueous layer was acidifiedto pH 1 with dil. hydrochloric acid and extracted with ethyl acetate.

The extract was washed with water, dried and evaporated. The residue wastriturated with ether to givebenzoyl-D-Ala-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3)(0.64 g).

I.R. (Nujol): 3290, 1720, 1680, 1640 cm⁻¹

(2) Step 2 ##STR264##

To a solution ofbenzoyl-D-Ala-L-ALa-γ-D-GLu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (0.58 g) in a mixture (30 ml) of methanol and water (2:1) was added10% palladium carbon and the mixture was shaken under three atmosphericpressure of hydrogen for 3 hours. The catalyst was filtered off and thefiltrate was evaporated. The residue was triturated with ether to givebenzol-D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4) (0.44 g).

I.R. (Nujol): 3250, 1720, 1660-1360 cm⁻¹.

(3) Step 3 ##STR265##

Benzoyl-D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4)(0.40 g) was dissolved in trifluoroacetic acid (6 ml) at 0° C. andthe solution was stirred at the same temperature for an hour. Afterevaporation of trifluoroacetic acid, a small amount of benzene was addedto the residue and the mixture was evaporated. The residue was dissolvedin water (3 ml) and the solution was acidified with 1N sulfuric acid(0.5 ml). To the solution was added dropwise a solution of sodiumperiodate (0.14 g) in water (3 ml) at 0° C. After stirring for an hour,sodium bisulfite was added thereto until the color of the reactionmixture became clear. After adjustment of the pH to 3.2, the reactionmixture was concentrated to about 4 ml. The concentrate was put on acolumn of a macroporous non-ionic adsorption resin, HP 20 (20 ml) and,after washing with water, eluted with 30% aqueous methanol. Thefractions containing the object compound (5) were concentrated and theconcentrate was lyophilized to givebenzoyl-D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (5)(120 mg).

N.M.R. (D₂ O), δ(ppm): 1.50 (3H, d, J=7 Hz), 1.57 (3H, d, J=7 Hz),1.42-2.42 (10H, m), 3.95 (2H, s), 3.37-4.67 (5H, m), 7.54-7.95 (5H, m).

The following compounds were prepared in substantially the same manneras that of Steps 1-3 of Example 27.

EXAMPLE 28

(1) Step 1

Acetyl-D-Ala-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.40 (18H, s), 1.33 (6H, d, J=7 Hz), 1.92 (3H,s), 1.84-2.30 (10H, m), 3.88 (2H, s), 3.84-4.25 (5H, m), 5.12 (2H, s),7.30 (5H, m).

(2) Step 2

Acetyl-D-Ala-L-Ala-γ-D-Glu(α-oBzl)-(L)-mesoDAP-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.32 (6H, d, J=7 Hz), 1.34-2.32 (10H, m), 1.90(3H, s), 3.79-4.25 (5H, m), 3.89 (2H, s), 5.12 (2H, s), 7.30 (5H, s).

(3) Step 3

Acetyl-D-Ala-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

N.M.R. (D₂ O), δ(ppm): 1.32 (3H, d, J=7 Hz), 1.40 (3H, d, J=7 Hz),1.25-2.38 (10H, m), 3.80 (1H, t, J=6 Hz), 3.96 (2H, s), 4.24-4.42 (4H,m).

EXAMPLE 29 ##STR266##

L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1) (662mg) was added to trifluoroacetic acid (4 ml) and the mixture was stirredat ambient temperature for 50 minutes. After removal of trifluoroaceticacid by evaporation, the residue was dissolved in water (20 ml) andcooled to 0° C. To this cooled solution were added 0.1N sulfuric acid(25 ml) and a solution of sodium periodate (535 mg) in water (5 ml). Themixture was stirred at the same temperature for an hour, during whichtime the reaction solution turned dark brown.

Sodium bisulfate was added until the color was clear and the mixture wasadjusted to pH 3 with dil. sodium bicarbonate. After concentration ofthe mixture, the residue was dissolved in a small amount of water andadjusted again to pH 3. The solution was put on a column of amacroporous non-ionic adsorption resin, HP 20 (130 ml) and eluted withwater. The fraction containing the object compound (2) was concentrated.This chromatograph operation was repeated, and the fraction containingthe object compound (2) was lyophilized to give an amorphous solid (220mg). A 180 mg portion of the solid was further purified by columnchromatograph using an active carbon (20 ml) and eluted with a mixtureof water and acetone (4:1). The eluate was evaporated and the residuewas pulverized with acetone to giveL-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (2) (90 mg).

[α]_(D) =-10.6 (C=0.34 water).

N.M.R. (D₂ O), δ(ppm): 1.55 (3H, d, J=7 Hz), 3.75 (1H, t, J=7 Hz), 3.86(2H, s), 4.05-4.45 (m).

EXAMPLE 30 ##STR267##

To a solution of Z-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (1) (2.04g) and sodium bicarbonate (890 mg) in a 50% aqueous dioxane (40 ml) wasadded ditertbutyldicarbonate (1.53 g). After stirring for 4 hours atambient temperature, additional ditertbutyldicarbonate (770 mg) 1Nsodium hydroxide (0.6 ml) was added thereto and the mixture was stirredfor 2.5 hours. To the mixture was added acetic acid (5 ml) and theresulting mixture was hydrogenated over 10% palladium black (300 mg)under an atmospheric pressure of hydrogen.

After the catalyst was filtered off, the filtrate was evaporated invacuo.

The residue was dissolved in water (5 ml) and put on a column ofmacroporous non-ionic adsorption resin, HP 20 (120 ml). The fractionseluted with a mixture of water and methanol (7:3) were combined andconcentrated to give L-ALa-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH(2)(1.17 g).

I. R. (Nujol): 3280, 1650 (shoulder), 1520 (shoulder).

N.M.R. (D₂ O), δ(ppm): 1.42 (9H, s), 1.55 (3H, d, J=7 Hz), 3.93 (2H, s),4.04-4.50 (4H, m).

EXAMPLE 31

(1) Step 1 ##STR268##

To a suspension ofbenzyloxycarbonyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(1)(4.43 g) in a mixture of 50% aqueous methanol was added 1N sodiumhydroxide (11 ml) at 0° C. After stirring for an hour at the sametemperature, the reaction mixture was concentrated to about 30 l ml,adjusted to pH 2 with 1N hydrochloric acid, and then extracted withethyl acetate (300 ml). The organic layer was washed with 0.5Nhydrochloric acid (30 ml) and brine, dried over magnesium sulfate andthen concentrated in vacuo to give a white powder which was washed withether. The powder was collected by filtration to givebenzyloxycarbonyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(2)(3.46 g).

N.M.R. (CD₃ OD), δ(ppm): 1.50 (18H, s), 3.98 (2H, s), 4.00-4.70 (4H, m),5.17 (2H, s), 7.40 (5H, s).

(2) Step 2 ##STR269##

Benzyloxycarbonyl-L-Al-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GLyOH(2)(3.30 g) was added to trifluoroacetic acid (15 ml) and the mixturewas allowed to stand at ambient temperature for 30 minutes. The mixturewas evaporated in vacuo and the residual oil was triturated with etherto give a powder (3.38 g). The solid was dissolved in water (60 ml) andto the solution was added 1N sulfuric acid and sodium periodate (1.78 g)at 0° C. After stirring at the same temperature for an hour, thereaction mixture was treated with sodium bisulfite, concentrated toabout 5 ml, adjusted to pH 2 with 1N sodium hydroxide and put on acolumn of macroporous non-ionic adsorption resin, HP 20 (400 ml). Thefraction eluted with a mixture of water and methanol (6:4) were combinedand evaporated in vacuo to givebenzyloxycarbonyl-L-Al-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (3)(1.03 g).mp 205°-207° C.

N.M.R. (D₂ O), δ(ppm): 1.37 (3H, d, J=7 Hz), 3.78 (1H, m), 3.93 (2H, s),4.05-4.40 (3H, m), 5.14 (2H, s), 7.45 (5H, s).

EXAMPLE 32

(1) Step 1 ##STR270##

Triethylamine (720 mg) and Z-L-Ala-D-Glu(oSu)oBzl (1)(3.51 g) was addedto a solution of Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH (2)(3.00 g) in amixture of dioxane (90 ml) and water (70 ml). The resulting mixture wasallowed to stand at ambient temperature for 18 hours, and concentratedto about 50 ml. The concentrate was adjusted to pH 3 with 1Nhydrochloric acid and extracted with ethyl acetate (300 ml) containingmethanol (20 ml). The organic layer was washed with brine (100 ml),dried over sodium sulfate and then evaporated in vacuo. The residue waswashed with ether and filtered to giveZ-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-GlyOH(3)(5.53 g).

N.M.R. (DMSO-d₆), δ(ppm): 1.07 (3H, d, J=7 Hz), 3.64-4.48 (6H, m), 5.04(2H, s), 5.12 (2H, s), 6.56 (1H, d, J=8 Hz), 7.35 (10H, s), 7.95 (1H, d,J=8 Hz), 8.15 (1H, m), 8.35 (1H, d, J=8 Hz), 8.70 (1H, s), 9.56 (1H, s).

(2) Step 2 ##STR271##

A solution of compound (3)(5.26 g) in acetic acid (120 ml) washydrogenated over 10% palladium black (2.0 g) under an atmosphericpressure of hydrogen. After the catalyst was filtered off and washedwith acetic acid, the filtrate and the washings were combined and thenevaporated in vacuo. The residual oil was triturated with ether to giveL-Ala-γ-D-Glu(α-OH)-(L)-Boc-D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (4)(3.90g).

I.R. (Nujol): 3250, 1660 (shoulder), 1515 cm⁻¹

N.M.R. (D₂ O), δ(ppm): 1.54 (3H, d, J=8 Hz), 3.88 (2H, s), 3.95-4.50(4H, m).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Example 1, respectively.

EXAMPLE 33

(1) Step 1

Diphenylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.42 (18H, s), 3.92 (2H, s), 3.83-4.52 (4H, m),5.08 (1H, s), 7.29 (10H, s).

(2) Step 2

Diphenylacetyl-L-Al-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

N.M.R. (D₂ O+NaHCO₃), δ(ppm): 1.40 (3H, d, J=7 Hz), 1.20-2.25 (10H, m),3.71 (1H, m), 3.80 (2H, s), 4.10-4.50 (3H, m),

5.17 (1H, s), 7.35 (10H, s).

EXAMPLE 34

(1) Step 1

N-(N-Benzyloxycarbonyl-L-5-oxo-2-pyrrolidinecarbonyl)-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

N.M.R. (CD₃ OD), δ(ppm): 1.47 (18H, s), 3.97 (2H, s), 5.27 (2H, s), 7.39(5H, s).

(2) Step 2

N-(N-Benzyloxycarbonyl-L-5-oxo-2-pyrrolidinecarbonyl)-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-55.0° (C=0.14, water).

N.M.R. (D₂ O), δ(ppm): 1.18 (3H, d, J=7 Hz), 1.3-2.8 (14H, m), 3.80 (1H,t, J=7 Hz), 3.94 (2H, s), 5.19 (2H, ABq), 7.39 (5H, s).

(3) Step 3

L-5-oxo-2-pyrrolidinecarbonyl-L-Al-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GLyOH.

N.M.R. (D₂ O), δ(ppm): 1.42 (3H, d, J=7 Hz), 3.79 (1H, t, J=7 Hz), 3.95(2H, s), 4.15-4.50 (4H, m).

EXAMPLE 35

(1) Step 1 ##STR272##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-β-ALaOH(3)was prepared in substantially5the same manner as the step 1 of Example12 from compound (1) and (2).

N.M.R. (DMSO-d₆) δ(ppm): 0.9-2.6(16H, m), 1.38(18H, S), 2.05(3H, S),2.9-4.7(8H, m), 5.00 (1H, q, J=7 HZ), 5.14 (2H, S), 6.5-6.7(1H, m),7.39(5H, S), 7.7-8.4(4H, m), 8.65(1H,broad S), 9.55(1H, broad S).

(2) Step 2 ##STR273##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-.beta.-AlaOH(4) was prepared in substantially the same manner as the step 2 ofExample 12 from compound (3).

N.M.R. (D₂ O), δ(ppm): 1.1-2.8(16H, m), 1.38(18 H, S), 2.55(2H, t, J=7HZ), 3.43(2H, t, J=7 HZ), 3.8-4.5 (5H, m).

(3) Step 3 ##STR274##

Di-trifluoroacetic acid salt ofD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-β-AlaOH (5) wasprepared in substantially the same manner the step 3 of Example 12 fromcompound (4).

N.M.R.(D₂ O), δ(ppm): 1.1-2.8(10H, m), 1.37(3H, d, J=7 HZ), 1.42(3H, d,J=7 HZ), 2.58(2H, t, J=6 HZ), 3.47(2H,t, J=6 HZ), 2.9-4.6(5H, m).

(4) Step 4 ##STR275##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-β-AlaOH (6) was prepared insubstantially the same manner as the step 4 of Example 12 for compound(5).

N.M.R. (D₂ O, δ(ppm): 1.37(3H, d, J=7 HZ), 1.44(3H, d, J=7 HZ),1.2-2.7(10H, m), 2.61(2H, t, J=7 HZ), 3.47(2H, t, J=7 HZ), 3.83(1H, t,J=6 HZ), 4.0-4.6(4H,m).

EXAMPLE 36

(1) Step 1 ##STR276##

D-Lac-(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-GluOH(3)was prepared in substantially the same manner as the step 1 of Example12 from compunds (1) and (2).

N.M.R. (DMSO-d₆), δ(ppm): 0.9-2.5(14H, m), 1.40(18H, S), 2.07(3H, S),3.6-4.6(5H, m), 4.99(1H, q, J=7 HZ), 5.15(2H, S), 6.5-6.9(1H, m),7.39(5H, S), 7.7-9.1(5H, m), 9.55(1H, broad S).

(2) Step 2 ##STR277##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-GluOH(4) was prepared in substantially the same manner as the step 2 ofExample 12 from compound (3).

N.M.R. (D₂ O), γ(ppm): 1.3-2.8(14H, m), 1.45(18H, S), 3.9-4.6(6H,m).

(3) Step 3 ##STR278##

Di-trifluoroacetic acid salt ofD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-D-GluOH (5) wasprepared in substantially the same manner as the step 3 of Example 12from compound (4).

N.M.R.(D₂ O), δ(ppm): 1.1-2.7 (m), 1.35(3H, d, J=7 HZ), 1.42(3H, d, J=7HZ), 3.9-4.6(6H, m).

(4) Step 4 ##STR279##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-GluOH (6) was prepared insubstantially the same manner as the step 4 of Example 12 from compound(5).

EXAMPLE 37

(1) Step 1 ##STR280##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-L-AlaOH(3) was prepared in substantially the same manner as the step 1 ofExample 12 from compounds (1) and (2).

N.M.R. (DMSO-D₆), δ(ppm): 1.0-2.4(19H, m), 1.43(18H, S), 2.10(3H, S),3.8-4.8(5H, m), 5.04(1H, q, J=7 HZ), 5.19(2H, S), 6.5-7.0(1H, m),7.42(5H, S), 7.7-8.9(5H, m), 9.61(1H, broad S).

(2) Step 2 ##STR281##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-L-AlaOH(4) was prepared in substantially the same manner as the step 2 ofExample 12.

N.M.R.(D₂ O), δ(ppm): 1.3-2.8(19H, m), 1.45(18H, S), 3.9-4.6(6H, m).

(3) Step 3 ##STR282##

Di-trifluoroacetic acid salt ofD-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-L-Ala(OH)(5)was prepared in substantially the same manner as the step 3 of Example12 from compound (4).

N.M.R. (D₂ O), δ(ppm): 1.2-2.7(10H, m), 1.39(3H, d, J=7 HZ), 1.44(6H, d,J=7 HZ), 3.9-4.7(6H, m).

(4) Step 4 ##STR283##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-L-Ala(OH) (6) was preparedin substantially the same manner as the step 4 of Example 12 fromcompound (5).

[α]_(D) =-41.0 (C=0.42, water),

N.M.R.(D₂ O), δ(ppm): 1.37(3H, d, J=7 HZ), 1.43(6H, d, J=7 HZ),1.2-2.5(10H, m), 3.8(1H, t, J=6 HZ), 4.1-4.5(5H, m).

EXAMPLE 38

(1) Step 1 ##STR284##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-Phe(OH)(3) was prepared in substantially the same manner as the step 1 ofExample 12 from compound (1) and (2).

NMR(DMSO-d₆), δ(ppm): 1.0-2.4 (16H, m), 1.39 (18H, s), 2.05 (3H, s),2.8-3.2 (2H, m), 3.7-4.7 (5H, m), 4.99 (1H, q, J=7 Hz), 5.16 (2H, s),6.65 (1H, broad d, J=8 Hz), 7.23 (5H, s), 7.39 (5H, s), 7.6-8.9 (5H, m),9.56 (1H, broad s).

(2) Step 2 ##STR285##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-PheOH(4) was prepared in substantially the same manner as step 2 of Example12 from compound (3).

I.R. (Nujol): 3270, 1720 (shoulder), 1645 (broad), 1520 (broad) cm⁻¹.

(3) Step 3 ##STR286##

Di-trifluoroacetic acid salt of D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-D-PheOH (5) was prepared insubstantially the same manner as the step 3 of Example 12 from compound(4).

N.M.R. (D₂ O), δ(ppm): 1.1-2.7(10H, m), 1.46(3H, d, J=7 Hz), 1.50 (3H,d, J=7 Hz), 2.8-3.5 (2H, m), 3.9-4.7 (6H, m), 7.41 (5H, s).

(4) Step 4 ##STR287##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-PheOH (6) was preparedin substantially the same manner as the step 4 of Example 12 fromcompound (5).

[α]_(D) =-28.6° (c=0.511 water).

N.M.R. (D₂ O), δ(ppm): 1.1-2.5 (10H, m), 1.36 (3H, d, J=7 Hz), 1.42 (3H,d, J=7 Hz), 3.14 (2H), 4.0-4.5 (5H, m), 7.1-7.5 (5H, m).

EXAMPLE 39

(1) Step 1 ##STR288##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-SarOH(3) was prepared in substantially the same manner as the step 1 ofExample 12 from compounds (1) and (2).

N.M.R. (DMSO-d6), δ(ppm): 1.0-2.4 (16H, m), 1.35 (18H, s), 2.03 (3H, s),2.80 and 3.05 (3H, a pair of singlets), 3.6-4.5 (6H, m), 4.98 (1H, q,J=7 Hz), 5.13 (2H, s), 6.5-7.0 (1H, m), 7.3 (1H, m), 7.35 (5H, s),7.7-9.0 (4H, m).

(2) Step 2 ##STR289##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-Sar OH(4) was prepared in substantially the same manner as the step 2 ofExample 12 from compound 3.

N.M.R. (D₂ O), δ(ppm): 1.1-2.5 (16H, m), 1.41 (9H, s), 1.43 (9H, s),2.93 and 3.18 (3H, a pair of singlets), 3.7-4.5 (7H, m).

(3) Step 3 ##STR290##

Di-trifluoroacetric acid salt ofD-LaC(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-Sar OH (5) wasprepared in substantially the same manner as step 3 of Example 12 fromCompound (4).

N.M.R. (D₂ O), (ppm): 1.0-2.6 (10H, m), 1.35 (3H, d, J=7 Hz), 1.41 (3H,d, J=7 Hz), 2.93 and 3.17 (3H, a pair of singlets), 3.8-4.6 (7H, m).

(4) Step 4 ##STR291##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-Sar OH (6) was prepared insubstantially the same manner as the step 4 of Example 12 from compound(5).

[α]_(D) =-37.6° (C=0.402, water).

N.M.R. (D₂ O), δ(ppm): 1.1-2.5 (10H, m), 1.35 (3H, d, J=7 Hz), 1.41 (3H,d, J=7 Hz), 2.96 and 3.20 (3H, a pair of singlet), 3.70-4.0 (1H, m),4.0-4.6 (6H, m).

EXAMPLE 40

(1) Step 1 ##STR292##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (3) was prepared in substantially the samemanner as step 1 of Example 12 from compounds (1) and (2).

NMR(DMSO-d₆), δ(ppm): 0.9-2.6(37H,m), 2.10(3H,s), 3.7-4.7(5H,m),5.01(1H,q,J=7 Hz), 5.19(2H,s), 6.6-7.0(1H,m), 7.42(5H,s),7.7-8.9(5H,m),9.60(1H,broad s).

(2) Step 2 ##STR293##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNH.Boc-(L)-(D)-AlaOH (4) was prepared in substantially thesame manner as the step 2 of Example 12 from compound (3).

NMR(D₂ O), δ(ppm): 1.3-2.7(19H,m), 1.48(18H,s), 3.9-4.7(6H,m).

(3) Step 3 ##STR294##

Di-trifluoroacetic acid salt of D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-D-AlaOH (5) was prepared in substantially the samemanner as the step 3 of Example 12 from compound (4).

NMR(D₂ O), δ(ppm): 1.36(3H,d,J=7 Hz), 1.41(6H,d,J=7 Hz), 1.1-2.7(10H,m),3.9-4.7(6H,m).

(4) Step 4 ##STR295##D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (6) was preparedin substantially the same manner as the step 4 of Example 12.

[α]_(D) =-17.4° (C=0.305, water).

NMR(D₂ O), δ(ppm): 1.38(3H,d,J=7 Hz), 1.41(3H,d,J=7 Hz), 1.44(3H,d,J=7Hz), 1.3-2.6(10H,m), 3.84(1H,t,J=6 Hz), 4.1-4.6(5H,m).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Example 1, respectively.

EXAMPLE 41

(1) Step 1 ##STR296##

N.M.R. (DMSO-d₆), δ(ppm): 0.80-2.40 (44H, m), 3.67-4.67 (4H, m), 6.70(1H, d, J=7 Hz), 7.83 (1H, d, J=7 Hz), 8.03 (2H, d, J=7 Hz), 8.60 (1H,s), 9.53 (1H, s)

(2) Step 2 ##STR297##

N.M.R. (D₂ O, δ(ppm): 0.84 (3H, t, J=7 Hz), 1.36 (3H, d, J=7 Hz),1.0-2.60 (20H, m), 3.80 (1H, t, J=7 Hz), 4.10-4.52 (3H, m).

EXAMPLE 42

(1) Step 1 ##STR298##

The compound (3) was prepared in substantially the same manner as thestep 1 of Example 12.

N.M.R. (DMSO-d₆), δ(ppm): 1.00-2.40 (10H, m), 1.24 (3H, t, J=7 Hz), 1.40(18H, s), 3.80-4.48 (4H, m), 5.04 (2H, s), 5.12 (2H, s), 6.74 (1H, d,J=7 Hz), 7.36 (10H, s), 8.04(1H, d, J=7 Hz), 8.32 (1H, d, J=7 Hz), 8.68(1H, broad s), 9.60 (1H, s).

(2) Step 2 ##STR299##

The compound (4) was prepared in substantially the same manner as thestep 2 of Example 12.

N.M.R. (D₂ O), δ(ppm): 1.36-2.66 (13H, m), 1.43 (18H, s), 3.83-4.43 (4H,m).

(3) Step 3 ##STR300##

The compound (5) was prepared in substantially the same manner as thestep 1 of Example 1.

N.M.R. (DMSO-d₆), δ(ppm): 1.25 (3H, d, J=7 Hz), 1.40 (18H, s), 1.30-2.40(10H, m), 3.83-4.50 (4H, m), 5.06 (2H, s), 6.76 (1H, d, J=7 Hz). 7.38(5H, s), 8.13 (2H, d, J=7 Hz), 8.70 (1H, broad s), 9.60 (1H, s).

(4) Step 4 ##STR301##

The compound (6) was prepared in substantially the same manner as thestep 4 of Example 12.

N.M.R. (D2O), δ(ppm): 1.34 (3H, d, J=7 Hz), 1.10-2.43 (10H, m) 3.71 (1H,t, J=7 Hz), 3.91-4.30 (3H, m), 5.10 (2H, s), 7.40 (5H, s).

(5) Step 5 ##STR302##

The compound (7) was prepared in substantially the same manner as theStep 2 of Example 11.

N.M.R. (D2O), δ(ppm): 1.52 (3H, d, J=7 Hz), 1.20-2.52 (10H, m), 3.76(1H, t, J=7 Hz), 4.00-4.40 (3H, m).

The following compounds were prepared in substantially the same manneras step 1 to 4 of Example 12, respectively.

EXAMPLE 43

(1) Step 1

D-Lac (oAc)-L-Ala-γ-D-Glu (α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc.

N.M.R. (DMSO-D6), δ(ppm): 1.1-2.5 (16H, m), 1.38(18H, s), 2.04 (3H, s),3.8-4.7 (4H, m), 4.95 (1H, q, J=7 Hz), 5.10 (2H, s), 7.30 (5H, s), 8.08(2H, broad t, J=8 Hz).

(2) Step 2

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc.

I.R. (Nujol): 1720 1650, 1525 cm⁻¹.

(3) Step 3

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ (trifluoroaceticacid salt).

N.M.R.(D₂ O), δ(ppm): 1.40 (3H, d, J=7 Hz), 1.45 (3H, d, J=7 Hz),1.2-2.7 (10H, m), 4.0-4.7 (5H, m).

(4) Step 4

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP.

[α]_(D) =-21.3° (c=0.258, water).

N.M.R. (D2O), δ(ppm): 1.1-2.5 (10H, m), 1.35 (3H, d, J=7 Hz), 1.42 (3H,d, J=7 Hz), 3.88 (1H, t, J=6 Hz), 4.0-4.5 (4H, m).

EXAMPLE 44

(1) Step 1

Benzyloxycarbonyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH.

I.R. (Nujol): 3290, 1720, 1650, 1630, 1530 cm⁻¹.

N.M.R. (CD₃ OD), δ (ppm): 1.39 (18H, s), 3.75-4.55 (5H, m), 5.06 (2H,s), 5.12 (2H, s), 7.23 (10H, s).

(2) Step 2

L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNH-Boc-(L)-D-AlaOH.

m.p. 174° (dec).

I.R. (Nujol): 3300, 1655, 1525 cm⁻¹.

N.M.R. (D₂ O), δ (ppm): 1.45 (18H, s), 3.98-4.60 (5H, m).

(3) Step 3

Heptanoyl L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-NHNHBoc-(L)-D-AlaOH.

I.R. (Nujol): 3300, 1720, (shoulder), 1650 cm⁻¹.

N.M.R. (DMSO-d₆), δ (ppm): 0.88 (3H, t, J=7 Hz), 1.0-2.4 (44H, m),4.0-4.6 (5H, m).

(4) Step 4

Heptanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH.

[α]_(D) =-26.0° (c=0.2, water).

I.R. (Nujol): 3250, 1720, 1640 cm⁻¹.

N.M.R. (D₂ O), δ (ppm): 0.86 (3H, t, J=7 Hz), 1.0-2.5 (26H, m), 3.84(1H, t, J=7 Hz), 4.2-4.56 (4H, m).

EXAMPLE 45

(1) Step 1

Acetoxyacetyl-L-Ala-γ-D-Glu-(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-NHNHBoc-(L)-D-AlaOH

I.R. (Nujol): 3280, 1720, 1650, 1520 cm⁻¹.

N.M.R. (CD₃ OD), δ (ppm): 1.47, (18H, s), 2.14 (3H, s), 4.07-4.70 (5H,m), 4.63 (2H, s).

(2) Step 2

Glycoloyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH.

I.R. (Nujol): 3280, 1720, 1630, 1530 cm⁻¹.

N.M.R. (D₂ O), δ (ppm): 1.41, (3H, d, J=7 Hz), 1.43 (3H, d, J=7 Hz),3.83 (1H, t, J=7 Hz), 4.14 (2H, s), 4.2-4.55 (4H, m).

EXAMPLE 46

(1) Step 1

O-Acetyl-D-Mandelyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH.

I.R. (Nujol): 3280, 1720, 1650, 1520 cm⁻¹.

N.M.R. (CD₃ OD), δ (ppm): 1.39, (18H, s), 2.15 (3H, s), 3.9-4.6 (5H, m),5.87 (1H, s), 7.38 (5H, s).

(2) Step 2

D-Mandelyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH.

I.R. (Nujol): 3270, 1715, 1640, 1520 cm⁻¹.

N.M.R. (D₂ O), δ (ppm): 1.35 (3H, d, J=7 Hz), 1.42 (3H, d, J=7 Hz),3.80, (1H, t, J=7 Hz), 4.1-4.5 (4H, m), 5.18 (1H, s), 7.32 (5H, s).

EXAMPLE 47

(1) Step 1

Phenylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH.

I.R. (Nujol): 3280, 1720, (shoulder), 1650, 1520 cm^(-I).

N.M.R. (CD₃ OD), δ (ppm): 1.40 (18H, s), 3.59 (2H, s), 3.94-4.50 (5H,m), 7.27 (5H, s).

(2) Step 2

Phenylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH.

I.R. (Nujol): 3250, 1715, 1640, 1530 cm⁻¹.

N.M.R. (D₂ O), γ (ppm): 1.36 (3H, d, J=7 Hz), 1.40 (3H, d, J=7 Hz), 3.65(2H, s), 3.80 (1H, t, J=7 Hz), 4.20-4.55 (4H, m), 7.36 (5H, s).

EXAMPLE 48

(1) Step 1

Benzyloxycarbonyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH.

I.R. (Nujol): 3270, 1715 (shoulder), 1650, 1520 cm⁻¹.

N.M.R. (CD₃ OD), δ (ppm): 1.41 (18H, s), 3.95-4.50 (5H, m), 5.07 (2H,s), 7.28 (5H, s).

(2) Step 2

Benzyloxycarbonyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH.

[α]_(D) =-57.4° (c=0.24, water).

I.R. (Nujol): 3250, 1700 (shoulder), 1640, 1520 cm⁻¹.

N.M.R. (D₂), δ (ppm): 1.40 (6H, d, J=8 Hz), 3.86 (1H, t, J=6 Hz),4.07-4.50 (4H, m), 5.10 (2H, s), 7.40 (5H, s).

EXAMPLE 49

(1) Step 1

Benzyloxycarbonyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-meso-DAP-(L)-GlyOH.

I.R. (Nujol): 3260, 1730-1630 (broad) cm⁻¹.

N.M.R. (DMSO-d₆), δ (ppm): 1.20-2.40 (10H, m), 1.40 (18H, s), 3.76 (2H,d, J=7 Hz), 4.0-4.5 (3H, m) 5.08 (2H, s), 5.15 (2H, s), 7.40 (10H, s).

(2) Step 2

γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3250 (broad), 1720-1620 (broad) cm⁻¹.

N.M.R. (D₂ O), δ (ppm): 1.20-2.70 (28H, m), 3.83 (1H, t, J=7 Hz), 3.94(2H, s), 4.0-4.5 (2H, m).

(3) Step 3

Heptanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

I.R. (Nujol): 3250, 1720 (shoulder), 1650 (broad) cm⁻¹.

N.M.R. (DMSO-d₆), δ (ppm): 0.86 (3H, t, J=7 Hz), 1.0-2.4 (38H, m), 3.70(2H, d, J=7 Hz), 4.0-4.5 (3H, m).

(4) Step 4

Heptanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

[α]_(D) =-18.0° (c=0.2, water).

I.R. (Nujol): 3300, 1720, 1640 (broad) cm⁻¹.

N.M.R. (D₂ O), δ (ppm): 0.84 (3H, t, J=7 Hz), 1.0-2.60 (20H, m), 3.80(1H, t, J=7 Hz), 3.96 (2H, s), 4.20-4.48 (2H, m).

EXAMPLE 50

(1) Step 1 ##STR303##

To a mixture of Boc-(D)-mesoDAP-(L)-D-AlaOH (1) (361 mg.) andtriethylamine (202 mg.) in 50% aqueous dioxane (20 ml.) was addedZ-D-Glu(OSu)OBZl (2) (440 mg.) and the reaction mixture was concentratedand washed with ethyl acetate. The organic layer was acidified withdiluted hydrochloric acid and extracted with ethyl acetate. The extractwas washed with water, dried over magnesium sulfate and evaporated togive a foamy residue, which was pulverized with isopropyl ether andcollected by filtration to give a white powder (520 mg.) ofZ-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (3).

I.R. (Nujol): 3300, 1720, 1690, 1640, 1530 cm⁻¹.

N.M.R. (DMSO-d₆), δ (ppm): 1.38 (9H, s), 1.00-2.66 (13H, m), 3.66-4.50(4H, m), 5.07 (2H, s), 5.15 (2H, s), 7.38 (10H, s).

(2) Step 2 ##STR304##

Z-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (3) (502 mg) wasdissolved in acetic acid (20 ml) and hydrogenated over 10%palladium-charcoal (100 mg). The catalyst was removed by filtration andthe filtrate was evaporated to give an oil, to which toluene was addedand evaporated to give a pasty residue. This residue was dissolved inwater and submitted to a column of HP-20 (50 ml), a macroporousnon-ionic adsorption resin. The column was eluted successively withwater and methanol-water (1:4). Evaporation of the latter fractions gavea white foam, which was dissolved in water and lyophillized to give awhite powder (290 mg) of γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(4).

I.R. (Nujol): 3250, 1720-1630 (broad), 1530 cm⁻¹.

N.M.R. (D₂ O), δ(ppm): 1.48 (9H, s), 1.10-2.66 (13H, m), 3.77 (1H, t,J=7 Hz), 4.00-4.66 (3H, m).

(3) Step 3 ##STR305##

To a mixture of γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (4) (268mg) and triethylamine (175 mg) in methanol (5 ml) was added a solutionof stearic anhydride (478 mg) in chloroform (10 ml) and the mixture waskept for 3 days at room temperature. The reaction mixture wasconcentrated and the residue was dissolved in ethyl acetate. The ethylacetate was washed successively with diluted hydrochloric acid andwater, dried over magnesium sulfate and evaporated to give a foamyresidue, which was pulverized with ether and collected to give a whitepowder (310 mg) ofstearoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (5).

I.R. (Nujol): 3300, 1710, 1695, 1660 1620 (broad), 1540 cm⁻¹.

N.M.R. (CD₃ OD), δ(ppm): 0.90 (3H, t, J=5 Hz), 1.33 (30H, s), 1.44 (9H,s), 1.16-2.66 (15H, m), 3.66-4.66 (4H, m).

(4) Step 4 ##STR306##

Stearoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (5) (308 mg) wasadded to trifluoroacetic acid (5 ml) and the mixture was stirred for 1hour at room temperature. The reaction mixture was concentrated andtriturated with ether to give a white powder, which was washed withether and methanol to givestearoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH(6) (230 mg.) as a whitepowder.

I.R. (Nujol: 3300, 1730, 1640, 1530 cm⁻¹.

N.M.R. (D₂ O+NaOD), δ(ppm): 0.86 (3H, t, J=6 Hz), 1.30 (30H, m),1.00-2.66 (15H, m), 3.21 (1H, t, J=7 Hz), 4.00-4.20 (3H, m).

EXAMPLE 51

(1) Step 1 ##STR307##

Compound (2)(4.75 g) was dissolved in a mixture of dioxane (30 ml) andwater (30 ml) containing triethylamine (1.01 g). To this solution, therewas added compound (1)(4.4 g) and the resulting solution was left for 1day at ambient temperature. The reaction mixture was concentrated andthe concentrates were subjected to extraction with ethylacetate. Theorganic layer was washed with water, dried over magnesium sulfate(MgSO₄) and then evaporated to give a formy residues, which werepulverised with diisopropylether and collected by filtration to givecompound (3)(5.77 g) as white powder.

I.R. (Nujol): 3270, 1730-1620 (broad).

N.M.R. (DMSO-d₆), δ(ppm): 1.37 (18H, s), 1.18-2.33 (13H, m), 3.66-3.84(4H, m), 5.06 (2H, s), 5.16 (2H, s), 7.36 (10H, s).

(2) Step 2 ##STR308##

Compound (3)(3.88 g) was dissolved in acetic acid (100 ml) andhydrogenated over palladium-charcoal (300 mg). Catalyst, i.e.palladium-charcoal was removed by filtration and the filtrate wasevaporated under reduced pressure to give an oily paste, to whichtoluene was added. The resulting solution was evaporated to give whitepowder residues, which were washed with diisopropylether and collectedby filtration to give compound (4)(2.10 g) as white powder.

I.R. (Nujol): 3300-2300 (broad), 1720-1620 (broad).

N.M.R. (D₂ O), δ(ppm): 1.47 (18H, s), 1.33-2.66 (13H, m), 3.66-4.50 (4H,m).

EXAMPLE 52

(1) Step 1 ##STR309##

Compound (1)(596 mg) was dissolved in methanol (30 ml), and to thissolution, there were added triethylamine (202 mg) and heptanoicanhydride (2)(363 mg) successively. The reaction mixture was kept for 3days at ambient temperature, and then concentrated. The concentrateswere subjected to extraction with ethylacetate. The organic layer waswashed with 10% aqueous hydrochloric acid and water, and dried overmagnesium sulfate (MgSO₄) and then evaporated to give a foamy residue,which was pulverized with ether and collected by filtration to givecompound (3)(470 mg) as white powder.

I.R. (Nujol): 3300, 1740-1730 (broad).

N.M.R. (DMSO-d₆), δ(ppm): 0.89 (3H, t, J=7 Hz), 1.00-2.40 (23H, m),3.66-4.66 (4H, m).

(2) Step 2 ##STR310##

Compound (3)(430 mg) was dissolved in trifluoroacetic acid (10 ml) andthe solution was kept for 30 minutes at ambient temperature. Thesolution was evaporated to give an oily paste, which was dissolved in0.1N-sulfuric acid (13 ml) and water (8 ml) and cooled in an ice-bath.To this solution, there was added aqueous NaIO₄ (278 mg in 5 ml water)under stirring, and the stirring was continued for 1 hour.

The resulting reaction mixture was treated with aqueous NaHSO₃ solutionuntil purple color of the solution disappeared. The pH of the solutionwas made neutral by addition of aqueous NaHCO₃ solution and the wholevolume of the solution was concentrated. The pH of the solution wasadjusted again to 2.0 and the whole solution was submitted to column ofHP-20 resin (50 ml). The column was eluted with water and thenmethanol-water (1:2). The latter fractions were evaporated gave a whiteformy substance, which was dissolved in water and then the solution waslyophillized to give compound (4)(240 mg) as white powder.

I.R. (Nujol): 3250, 1740, 1666-1640 (broad).

N.M.R. (D₂ O), δ(ppm): 0.82 (3H, t, J=6 Hz), 1.00-2.60 (20H, m), 1.35(3H, d, J=8 Hz), 3.81 (1H, t, J=7 Hz), 4.40-4.80 (3H, m).

EXAMPLE 53

(1) Step 1 ##STR311##

Compound (1)(596 mg) was dissolved in methanol (30 ml) and chloroform (5ml), and to this solution, there were added triethylamine (202 mg) andlauric anhydride (2)(609 mg) successively. The reaction mixture wastreated substantially in the same manner as the Step 1 of Example 52 togive compound (3)(570 mg) as white powder.

I.R. (Nujol): 3250, 1725-1610 (broad).

N.M.R. (DMSO-d₆), δ(ppm): 0.84 (3H, t, J=7 Hz), 1.30 (18H, s), 1.00-2.40(33H, m), 3.80-4.40 (3H, m).

(2) Step 2 ##STR312##

Compound (3)(510 mg) was dissolved in trifluoroacetic acid (10 ml) andthe solution was kept for 30 minutes at ambient temperature. Hereafter,the reaction mixture was treated substantially in the same manner as theStep 2 of Example 52 to give compound (4)(250 mg) as white powder.

I.R. (Nujol): 3300, 1720, 1660-1620 (broad).

N.M.R. (CD₃ OD), δ(ppm): 0.90 (3H, t, J=7 Hz), 1.00-2.60 (33H, m), 3.75(1H, t, J=7 Hz), 4.20-4.80 (3H, m).

EXAMPLE 54

(1) Step 1 ##STR313##

Compound (1)(596 mg) was dissolved in methanol (30 ml), and to thissolution, there were added triethylamine (202 mg) and manderic anhydride(2)(267 mg) successively. The reaction mixture was treated substantiallyin the same manner as the Step 1 of Example 52 to give Compound (3)(330mg) as white powder.

IR(Nujol: 3300, 1730-1640 (broad).

NMR(DMSO-d₆), δppm: 1.39(18H,s), 1.16-2.33(13H,m), 3.70-4.50(4H,m),5.00(1H, broad s), 7.40(5H, broad s).

(2) Step 2 ##STR314##

Compound (3) (479 mg) was dissolved in trifluoroacetic acid (10 ml) andthe solution was kept for 30 minutes at ambient temperature. Hereafter,the reaction mixture was treated substantially in the same manner as theStep 2 of Example 53 to give Compound (4) (190 mg) as white powder.

IR(Nujol): 3250, 1720, 1660-1620(broad).

NMR(D₂ O), δppm: 1.36(3H,d,J=7 Hz), 1.20-2.60 (10H,m), 3.80(1H,t,J=6Hz), 4.10-4.60 (3H,m), 5.20(1H,s), 7.42(5H,s).

EXAMPLE 55

(1) Step 1 ##STR315##

Compound (1)(1.51 g) was dissolved in a mixture of methanol (15 ml) andwater (10 ml). To this solution, there was added 1N-NaOH solution (4.0ml) and the solution was kept for 2 hours at ambient temperature. Thereaction mixture was concentrated after adjusting the pH of the solutionto 7.0. The residual aqueous solution was diluted with water. Afterextraction with ether, aqueous layer was acidified to pH 2 with 1N-HClto separate an oily product, which was extracted with ethylacetate. Theorganic layer was washed with H₂ O and dried over MgSO₄. Evaporation ofthe solvent gave a white foam, which was pulverized with diisopropylether to give Compound (2)(1.16 g) as white powder.

I.R. (Nujol): 3250, 1740-1620 (broad).

N.M.R. (DMSO-d₆), δ(ppm): 1.36 (18H, s), 1.00-2.40 (13H, m), 3.66-4.50(4H, m), 5.03 (2H, s), 7.36 (5H, s).

(2) Step 2 ##STR316##

Compound (2) (1.03 g.) was dissolved in trifluoroacetic acid (10 ml.)and the solution was kept for 30 minutes at ambient temperature.Hereafter, the reaction mixture was treated substantially in the samemanner as the Step 2 of Example 2 to give Compound (3) (0.6 g.) as whitepowder.

I.R. (Nujol): 3300, 1700, 1660-1620 (broad).

N.M.R. (D₂ O), δ(ppm) 1.36 (3H, d, J=7 Hz), 1.20-2.60 (10H, m), 3.76(1H, t, J=7 Hz), 3.96-4.50 (3H, m), 5.08 (2H, s), 7.40 (5H, s).

(3) Step 3 ##STR317##

Compound (3) (374 mg.) was dissolved in acetic acid (30 ml.) andhydrogenated over palladium-charcoal (100 mg.). Catalyst was removed byfiltration and the filtrate was evaporated under reduced pressure togive an oily paste, to which toluene was added. Evaporation of toluenegave white powder residue. The residue was dissolved in water andsubmitted to column of HP-20 resin (100 ml.). The column was eluted withwater. Evaporation of the fractions containing the product wasevaporated to give a pasty residue, which was dissolved in H₂ O and thesolution was lyophillized to give Compound (4) (200 mg.) as whitepowder.

I.R. (Nujol): 3250-2500 (broad) 1680-1500 (broad).

N.M.R. (D₂ O), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.20-2.60 (10H, m), 3.75(1H, t, J=7 Hz), 3.80 (1H, t, J=7 Hz), 4.00-4.50 (2H, m).

EXAMPLE 56

(1) Step 1 ##STR318##

Compound (1) was dissolved in dichloromethane (20 ml.) andN-methyl-morpholine (308 mg.) was added. This solution was cooled in adry ice-acetone bath (-17°--19° C.) under stirring, and to the solution,isobutylchlorocarbonate (417 mg.) was added. The reaction mixture wasallowed to react for 20 minutes at the same temperature. To theresulting reaction mixture was added to the solution of Compound (2)(1.41 g.) in a mixture of dichloromethane (40 ml.) andN,N-dimethylformamide (4 ml) containing bis(trimethylsilyl)acetamide (3ml.). The reaction mixture was stirred for 2 hours at -18° C.-10° C. andconcentrated under reduced pressure to give an oily residue, which wasdissolved in ethylacetate (100 ml.) and washed with 0.5N-Hcl. Theorganic layer was washed with brine and dried over MgSO₄. Evaporation ofthe solvent gave a white foam which was throughly washed with ether toafford Compound (3) (2.42 g.) as white powder, mp. 145°-149° C.(decomp.).

I.R. (Nujol): 3280, 1780. 1720. 1650.

N.M.R. (CD₃ OD), δ(ppm): 3.93 (2H, s), 5.18 (4H, s), 7.38 (10H, s).

(2) Step 2 ##STR319##

To a solution of Compound (3) (2.36 g.) in methanol (25 ml.), there wasadded 1N-NaOH (5.6 ml.) and the mixture was stirred for 5 hours atambient temperature. The reaction mixture was concentrated in vacuum,and to the residue, there was added 1N-HCl (6 ml.) and the solution wassubjected to extraction with ethylacetate. The organic layer was washedwith 0.5N-HCl (30 ml.), dried over MgSO₄ and evaporated in vacuum. Theresidual oil was treated with diethyl ether to give Compound (4) (1.86g.) as white powder.

I.R. (Nujol): 3300, 1680 (broad), 1625.

N.M.R. (CD₃ OD), δ(ppm): 1.46 (18H, s), 3.95 (2H, s), 4.00-4.50 (4H, m),5.13 (2H, s), 7.45 (5H, s).

(3) Step 3 ##STR320##

Compound (4) (1.8 g.) was added to trifluoroacetic acid (10 ml.) and themixture was stood for 100 minutes at ambient temperature. The reactionmixture was evaporated and the residue was triturated with ethyl etherto give a solid. The solid was dissolved in water (45 ml). To thissolution, was added 1N-H₂ SO₄ (4.4 ml.) and NaIO₄ (930 mg.) at 0° C.After stirring for 2 hours at 0° C., the reaction mixture was treatedwith aqueous NaHSO₃, concentrated to about 10 ml, adjusted to pH 2 with1N-NaOH and submitted to column of HP-20 resin (200 ml.). The column waseluted with water-methanol (3:2) and the fractions were combined andevaporated, and then the residue was lyophilized to give Compound (5)(680 mg.).

I.R. (Nujol): 3270, 1700, 1640, 1520.

N.M.R. (D₂ O), δ(ppm): 1.3-2.6 (14H, m), 3,83 (1H, t, J=7 Hz), 3.95 (2H,s), 4.05-4.50 (3H, m), 5.11 (2H, s), 7.40 (5H, s).

(4) Step 4 ##STR321##

A solution of Compound (5) (550 mg.) in water (6 ml.) was hydrogenatedover 10% palladium-carbon (180 mg.) under an atmospheric pressure ofhydrogen gas. After the catalyst was filtered off, the filtrate wasevaporated in vacuum and lyophilized to give Compound (6)(450 mg).

I.R. (Nujol): 3250, 1720 (shoulder), 1630, 1530.

N.M.R. (D₂ O), δ(ppm): 1.3-2.6 (14H, m), 3.74 (1H, t, J=6 Hz), 3.85 (2H,s), 3.99-4.50 (3H, m).

EXAMPLE 57

(1) Step 1 ##STR322##

To a mixture of Compound (1) (674 mg.) and N-methylmorpholine (202 mg.)in dichloromethane (30 ml.), there was added isobutyl chloroformate (273mg.) at -15°--20° C. and the mixture was stirred for 30 minutes at thesame temperaure. The mixture was cooled to -40° C., and to this, therewas added trimethyl silyl ester of Compound (2), which was prepared fromCompound (2) (910 mg.) and bis(trimethylsilyl)acetamide (3 ml.) bystirring in a mixture of dichloromethane (20 ml.) andN,N-dimethylformamide (2 ml.). This mixture was stirred for 1 hour at-15°--20° C., and after evaporation of the solvent, the residue wasdissolved in ethylacetate (100 ml.), and then the solution was washedsuccessively with 2%--HCl and water, dried over MgSO₄ and evaporated.The residue was pulverized with isopyropyl ether to give Compound (3)(1.25 g.).

N.M.R. (CDCl₃), δ(ppm): 1.43 (27H, s), 1.3-2.4(10H, m), 3.8-4.5 (5H, m),5.13 (2H, s), 7.33 (5H, s).

(2) Step 2 ##STR323##

To a solution of Compound (3) (1.08 g.) in 50% aqueous methanol (10 ml.)was added 1N-NaOH (3 ml.) and the mixture was stirred for 3 hours atambient temperature. The reaction mixture was neutralized with 1Nhydrochloric acid (3 ml.) and evaporated. The residue was dissolved intrifluoroacetic acid (5 ml.) and stirred for 15 minutes at ambienttemperature. After evaporatuion of trifluoroacetic acid, the residue waspulverized with diethyl ether. The powder thus obtained was dissolved inwater (10 ml.), and 1N hydrochloric acid (1.7 ml.) was added thereto. Tothis solution, was added a solution of NaIO₄ (354 mg.) in water (2 ml.)at 0° C., and the mixture was stirred for 1 hour at the sametemperature. After excess of NaIO₄ was decomposed by adding sodiumsulfite, the reaction mixture was adjusted to pH 4 with 1N aqueoussolution of sodium hydroxide and then concentrated to about 3 ml. Theconcentrate was subjected to column chromatography on non-ionicadsorption resin "Diaion HP-20: (Trade Mark, manufactured by MitsubishiChemical Industries Ltd.) (60 ml.) and elution was carried out withwater. The fractions containing the object compound was lyophilized togive compound (4) (140 mg.).

I.R. (KBr): 3500-2500, 1720, 1650.

N.M.R. (D₂ O), δ(ppm): 1.3-2.7 (10H, m), 3.90-4.50(3H, m), 4.00 (2H, s).

EXAMPLE 58

(1) Step 1 ##STR324##

To a mixture of Compound (1) (560 mg.) and N-methylmorpholine (202 mg.)in dichloromethane (30 ml.), there was added isobutyl chloroformate (273mg.) at -15°--20° C. and the mixture was stirred for 30 minutes at thesame temperature. The mixture was cooled to -40° C. and, to this, therewas added trimethylsilyl ester of Compound (2), which was prepared fromCompound (2) (910 mg.) and bis(trimethylsilyl)acetamide (3 ml.) bystirring in a mixture of dichloromethane (20 ml.) andN,N-dimethylformamide (2 ml.). The mixture was stirred for 1 hour at-15°--20° C., and then treated in the same manner as the Step 1 ofExample 57 to give Compound 3 (1.8 g.).

I.R. (Nujol): 3300, 1740, 1660.

N.M.R. (CD₃ OD), δ(ppm): 1.58(18H, s), 1.95 (3H, s), 1.50-2.50 (10H, m),3.88 (2H, s), 3.8-4.5 (3H, m).

(2) Step 2 ##STR325##

Compound (3) (0.95 mg.) was added to trifluoroacetic acid (5 ml.) andthe solution was stirred for 15 minutes at ambient temperature. Aevaporation of CF₃ COOH, the residue was dissolved in water (10 ml) and1N-HCl (1.7 ml) was added thereto. This solution was cooled to 0° C. anda solution of NaIO₄ (364 mg) in water (5 ml) was added thereto and themixture was stirred for 1 hour at the same temperature. An excess of thereagent was decomposed, by adding Na₂ SO₃, and then the mixture wasneutralized to pH 3 with 1N-NaOH and submitted to column of HP-20 (20ml.). The column, after washing with H₂ O, was eluted withmethanol:water (1:1). The fractions were concentrated, and the residuewas dissolved in 50% aq. acetic acid (10 ml.) and hydrogenated over 10%palladium carbon (100 mg). After removal of the catalyst, the filtratewas concentrated. The residue was dissolved in H₂ O and evaporated inorder to remove a trace of acetic acid. This operation was repeatedfurther two times. The residue was lyophilized to give Compound (4)(200mg).

I.R. (Nujol): 3250, 1715, 1640.

N.M.R. (D₂ O), δ(ppm): 1.5-2.1 (10H, m), 2.1 (3H, s), 3.8-4.5 (3H, m),4.0 (2H, s).

EXAMPLE 59

(1) Step 1 ##STR326##

To a cooled mixture of Compound (1) (2.43 g.) and N-methylmorpholine(0.71 g.) in methylene chloride (40 ml.) was added dropwise isobutylchloroformate (960 mg.) and the mixture was stirred for 20 minutes at 0°C. To this mixture was added a solution of Compound (2) (2.83 g.) in amixture of methylene chloride (20 ml.) and dimethylformamide (10 ml.)containing bis(trimethylsilyl)acetamide (2 ml.). The reaction mixturewas stirred for 2 hours and then concentrated to give an oily residue,which was dissolved in ethyl acetate, washed with water and dried overmagnesium sulfate. Evaporation of the solvent gave a white foam, whichwas pulverized with isopropyl ether to give Compound (3) (4.20 g.) aswhite powder.

I.R. (Nujol): 3300, 1740-1640 (broad) cm⁻¹.

N.M.R. (DMSO-d6), δ(ppm): 1.37 (18H, s), 1.17-2.40 (10H, m), 3.70-4.40(3H, m), 5.03 (2H, s), 5.17 (2H, s), 7.37 (10H, s).

(2) Step 2 ##STR327##

To a solution of Compound (3) (1.46 g.) in 50% aqueous methanol (10 ml.)was added 1N sodium hydroxide (4.10 ml.) and the mixture was stirred for2 hours at room temperature. The reaction mixture was concentrated andthe resulting aqueous solution was diluted with water. After washingwith ether, the aqueous layer was acidified with 1N hydrochloric acid topH 2 and extracted with ethyl acetate. The extract was washed withwater, dried over magnesium sulfate to give a white foam, which waspulverized with isopropyl ether to give Compound (4) (1.20 g.) as whitepowder.

I.R. (Nujol): 3400, 1740 1650 (broad) cm⁻¹.

N.M.R. (DMSO-d6), δ(ppm): 1.37 (18H, s), 1.17-2.40 (10H, m), 3.70-4.33(3H, m), 5.03 (2H, s), 7.37 (5H, s).

(3) Step 3 ##STR328##

Compound (4) (1.05 g.) was dissolved in trifluoroacetic acid (5 ml.) andstirred for 15 minutes at room temperature. Evaporation of the solventgave an oily residue, to which toluene was added and evaporated. Theresidue was dissolved in 0.1N sulfuric acid (35 ml.) and cooled in anice-bath. To this solution was added a solution of sodium periodate (640mg.) in water (5 ml.) and the mixture was stirred for 1 hour at the sametemperature. The reaction mixture was treated with aqueous sodiumbisulfite solution until the dark brown color of the solution wasdisappeared. After adjusting the pH of the solution to 4.0, the solutionwas concentrated to about 4 ml. and the pH was adjusted to 2.0. Thissolution was applied to a column of HP-20 (60 ml.) and eluted withwater. Subsequent elution with 50% aqueous methanol gave fractionscontaining the object product, which were combined and evaporated togive a pasty residue. This residue was dissolved in water andlyophillized to give Compound (5) (0.453 g.) as white powder.

N.M.R. (D₂ O), δ(ppm): 1.17-2.70 (10H, m), 3.83 (1H, t, J=7 Hz),4.0-4.60 (2H, m), 5.13 (2H, s), 7.43 (5H, s).

(4) Step 4 ##STR329##

Compound (5) (0.40 g.) was dissolved in water (20 ml.) and hydrogenatedover 10% palladium-charcoal (50 mg.). The catalyst was removed byfiltration and the filtrate was applied to a column of HP-20 (50 ml.)and eluted with water. Evaporation of the fractions containing theobject compound gave a pasty residue, which was dissolved in water andlyophillized to give Compound (6) (260 mg.) as white powder.

I.R. (Nujol): 3500-2500 (broad), 1710 (shoulder), 1620 cm⁻¹.

N.M.R. (D₂ O), δ(ppm): 1.20-2.60 (10H, m), 3.60-3.92 (2H, m), 4.0-4.20(1H, m).

EXAMPLE 60

(1) Step 1 ##STR330##

Compound (1) (2.30 g.) was dissolved in acetic acid (40 ml.) andhydrogenated over 10%-palladium-charcoal (300 mg.). The catalyst wasremoved by filtration and the filtrate was evaporated under reducedpressure to give an oily paste, to which toluene was added. Evaporationof toluene afforded white powder residue, which was throughly washedwith diisopropyl ether and collected by filtration to give Compound (2)(1.70 g.) as white powder.

I.R. (Nujol): 3500-2500 (broad), 1740-1650 (broad).

N.M.R. (D₂ O), d(ppm): 1.50 (18H, s), 1.16-2.83 (10H, m), 3.83 (1H, t,J=7 Hz), 4.0-4.50 (2H, m).

(2) Step 2 ##STR331##

Compound (2) (800 mg.) was dissolved in methanol (10 ml.) and heptanoicanhydride (380 mg.) was added thereto. The reaction mixture was keptovernight at ambient temperature. Evaporation of the solvent gave anoily paste, which was triturated with diisopropyl ether to give Compound(3) (800 mg.) as white powder.

I.R. (Nujol): 3300, 1740-1650 (broad).

N.M.R. (DMSO-d6), δ(ppm): 0.83-2.40 (41H, m), 3.83-4.50 (3H, m).

(3) Step 3 ##STR332##

Compound (3) (750 mg.) was dissolved in trifluoroacetic acid (4 ml.) andkept for 15 minutes at ambient temperature. Evaporation oftrifheoroacetic acid gave an oily paste, which was suspended in toluene.Evaporation of toluene afforded white foamy residue, which was dissolvedin 0.1N-H₂ SO₄ (30 ml.) and cooled in an ice bath. To this stirredsolution, there was added aqueous NaIO₄ (350 mg. in 10 ml. H₂ O) andstirring was continued for 1 hour. The resulting reaction mixture wastreated with aqueous NaHSO₃ solution until the purple color of thesolution disappeared. pH of the solution was made neutral by addingsolid NaHCO₃ and the whole volume was concentrated to about 5 ml. pH ofthe solution was adjusted again to 2.0 and the whole solution wassubmitted to Column of HP-20 resin (100 ml.). The column was eluted withwater and then Methanol-water (4:6). Evaporation of the latter fractionscollected gave white foam, which was dissolved in water and lyophilizedto give Compound (4) (180 mg.) as white powder.

I.R. (Nujol): 3300-2400 (broad), 1720, 1630.

N.M.R. (D₂ O), δ(ppm): 0.84 (3H, t, J=7 Hz), 1.0-2.60 (20H, m), 3.80(1H, t, J=7 Hz), 4.20-4.40 (2H, m).

EXAMPLE 61

(1) Step 1 ##STR333##

To a mixture of Boc-(D)-mesoDAP-(L)-D-AlaOH (1) (3.40 g) andtriethylamine (1.90 g) in 50% aqueous dioxane (80 ml) was addedZ-L-Ala-D-Glu(γ-OSu)OBzl (2) (5.10 g) and the mixture was left overnightat room temperature. After evaporation of dioxane, the remaining aqueoussolution was acidified with 1N hydrochloric acid and extracted withethyl acetate. The extract was washed with water, dried over magnesiumsulfate and evaporated to giveZ-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (3) (7.30 g) aswhite foam.

I.R. (Nujol): 3300, 2600-2400, 1710, 1650 cm⁻¹.

N.M.R. (DMSO-d₆), δ: 1.0-2.50 (25H, m), 3.80-4.50 (5H, m), 5.05 (2H, S),5.15 (2H, S), 7.40 (10H, S).

(2) Step 2 ##STR334##

A solution of Z-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(3) (7.0 g) in acetic acid (60 ml) was hydrogenated over 10%palladium-charcoal (0.50 g). The catalyst was removed by filtration andthe filtrate was evaporated to give a white foam, which was dissolved inwater (40 ml) and applied to a column of HP-20 (150 ml). The column waswashed with water and eluted with methanol-water (3:7). The eluate wasconcentrated and lyophillized to giveL-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (4) (4.30 g) aswhite powder.

I.R. (Nujol): 3300, 2600-2400, 1720 (shoulder), 1660 (broad) cm⁻¹.

N.M.R. (D₂ O), δ: 1.20-2.50 (10H, m), 1.40 (9H, S), 1.36 (3H, d, J=7Hz), 1.54 (3H, d, J=7 Hz), 3.90-4.50 (4H, m).

(3) Step 3 ##STR335##

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-ALaOH (5) wasprepared from L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (4)and stearic anhydride in substantially the same manner as that of Step 1of Example 1.

I.R. (Nujol): 3300, 1710, 1635, 1520 cm⁻¹.

N.M.R. (CD₃ OD-CDCl₃), δ: 0.90 (3H, t, J=7 Hz), 1.33 (30H, S), 1.43 (9H,S), 1.00-2.66 (18H, m), 3.60-4.60 (5H, m).

(4) Step 4 ##STR336##

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (6) was preparedfrom Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (5) insubstantially the same manner as that of Step 2 of Example 1.

I.R. (Nujol): 3300, 1730, 1650-1620, 1530 cm⁻¹.

N.M.R. (D₂ O+NaOD), δ: 0.90 (3H, t, J=7 Hz), 1.00-2.66 (51H, m), 3.24(1H, t, J=7 Hz), 4.00-4.66 (4H, m).

EXAMPLE 62

(1) Step 1 ##STR337##

Z-D-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (3)was prepared from Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (1) andZ-D-Ala-D-Glu(γ-OSu)OBzl (2) in substantially the same manner as that ofStep 1 of Example 1.

I.R. (Nujol): 3300, 2600 (shoulder), 1730, 1670, 1630 cm⁻¹.

N.M.R. (DMSO-d₆), δ: 1.00-2.60 (34H, m), 3.88-4.40 (5H, m).

(2) Step 2 ##STR338##

D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (4) wasprepared fromZ-D-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBOC-(L)-D-AlaOH (3)in substantially the same manner as that of Step 2 of Example 61.

I.R. (Nujol): 3300, 2600-2400 (shoulder), 1720 (shoulder), 1660 cm⁻¹.

N.M.R. (D₂ O), δ: 1.00-2.60 (34H, m), 3.88-4.40 (5H, m).

(3) Step 3 ##STR339##

To a cooled solution ofD-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (4)(1.35 g) in 50% aqueous acetone (10 ml) was added IN sodium hydroxide.O-Acetyl-D-mandelyl chloride (0.61 g) was added dropwise at 0° C.,during which time the pH was maintained at 8.0-9.0. After stirring for 1hour at the same time, the reaction mixture was adjusted to pH 7.0 withIN hydrochloric acid and acetone was evaporated. The resulting aqueoussolution was adjusted to pH 2.0 with IN hydrochloric acid and extractedwith ethyl acetate. The extract was washed with water, dried overmagnesium sulfate and concentrated to give a residue, which waspulverized with ether to giveO-acetyl-D-mandelyl-D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(5) (1.35 g) as a solid.

I.R. (Nujol): 3300, 1720, 1650, 1520 cm⁻¹.

N.M.R. (CD₃ OD), δ: 1.43 (18H, S), 1.00-2.66 (16H, m), 2.15 (3H, S),4.00-5.00 (5H, m), 5.95 (1H, S), 7.35 (5H, S).

(4) Step 4 ##STR340##

To a solution ofO-acetyl-D-mandelyl-D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(6) (1.25 g) in a mixture of methanol (20 ml) and water (10 ml) wasadded IN sodium hdyroxide (4.5 ml). After stirring the mixture for 1hour at room temperature, the pH of the mixture was adjusted to 6.0.After evaporation of methanol, the resulting aqueous solution wasadjusted to pH 2.0 with IN hydrochloric acid and extracted with ethylacetate. The extract was washed with water, dried over magnesium sulfateand concentrated. The residue was reached with ether to giveD-mandelyl-D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(6) (0.96 g) as a solid.

I.R. (Nujol): 3280, 1720-1650, 1520 cm⁻¹.

N.M.R. (CD₃ OD), δ: 1.43 (18H, S), 1.00-2.66 (16H, m), 3.83-4.66 (5H,m), 5.10 (1H, S), 7.17-7.66 (5H, m).

(5) Step 5 ##STR341##

D-Mandelyl-D-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (7) was preparedfromD-mandelyl-D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(6) in substantially the same manner as that of Step 2 of Example 1.

I.R. (Nujol): 3280, 1720, 1630-1610, 1520 cm⁻¹.

N.M.R. (D₂ O), δ: 1.32 (3H, d, J=7 Hz), 1.40 (3H, d, J=7 Hz), 1.20-2.40(10H, m), 3.74 (1H, t, J=6 Hz), 4.00-4.50 (4H, m), 5.18 (1H, S), 7.42(5H, S).

EXAMPLE 63

(1) Step 1 ##STR342##

Heptanoyl-D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBOC-(L)-D-AlaOH(2) was prepared fromD-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (1) insubstantially the same manner as that of Step of 1 of Example 1.

I.R. (Nujol): 3250, 1720, 1660-1640, 1530 cm⁻¹.

N.M.R. (CD₃ OD), δ: 0.90 (3H, t, J=7 Hz), 1.00-2.60 (44H, m), 3.90-4.60(5H, m).

(2) Step 2 ##STR343##

Heptanoyl-D-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (3) was preparedfrom heptanoyl-D-Ala-γ-D-Glu(α-OH)-(L)Boc-(D)-mesoDAP-(L)-D-AlaOH (2) insubstantially the same manner as that of Step 2 of Example 1.

I.R. (Nujol): 3300, 1720, 1620, 1520 cm⁻¹.

N.M.R. (D₂ O), δ: 0.80 (3H, t, J=6 Hz)., 1.36 (6H, d, J=7 Hz), 1.00-2.60(18H, m), 3.76 (1H, t, J=7 Hz), 4.00-4.50 (4H, m).

EXAMPLE 64

(1) Step 1 ##STR344##

Z-L-Ala-γ-D-Glu(α-OBzl)-(L)Boc-(D)-mesoDAP-(L)-GlyOH (3) was preparedfrom Boc-(D)-mesoDAP-(L)-GlyOH-(1) and Z-L-Ala-(D)Glu(α-Osu)OBzl (2) insubstantially the same manner as that of Step 1 of Example 61.

I.R. (Nujol): 3300, 2600-2400, 1720, 1690, 1650 cm⁻¹.

N.M.R. (DMSO-d₆), δ(ppm): 1.30 (3H, d, J-7 Hz), 1.10-2.50 (19H, m), 3.80(2H, d, J=7 Hz), 3.80-4.50 (4H, m), 5.05 (2H, s), 5.18 (2H, s), 7.40(10H, s).

(2) Step 2 ##STR345##

L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH (4) was prepared fromZ-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH (3) insubstantially the same manner as that of Step 2 of Example 61.

I.R. (Nujol): 3300, 2600-2400 (broad), 1720, 1650, (broad) cm⁻¹.

N.M.R. (D₂ O), δ(ppm): 1.56 (3H, d, J-7 Hz), 1.40 (9H, s), 1.20-2.50(10H, m), 3.92 (2H, s), 4.0-4.40 (4H, m).

(3) Step 3 ##STR346##

Arachidonyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(L)-GlyOH (5) wasprepared from L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GluOH (4) andN-hydroxyphthalimido ester of arachidonic acid in substantially the samemanner as that of Step 3 of Example 61.

I.R. (Nujol): 3300, 1720, 1640, 1530 cm⁻¹.

N.M.R. (DMSO-d₆), δ(ppm): 0.83 (3H, m), 1.1-2.9 (42H, m), 3.82 (2H, s),4.05-4.50 (4H, m), 5.20-5.50 (8H, m).

(4) Step 4 ##STR347##

Arachidonyl-L-Ala-γ-D-Glu(γ-OH)-(L)-mesoDAP-(L)-GlyOH (6) was preparedfrom arachidonyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH (5)in substantially the same manner as the Step 2 of Example 1.

I.R. (Nujol): 3270, 1720, 1635, 1530 cm⁻¹.

N.M.R. (D₂ O=NaOD), δ(ppm): 0.85 (3H, m), 1.1-2.9 (33H, m), 3.23 (1H, t,J=7 Hz), 3.76 (2H, s), 4.1-4.5 (3H, m), 5.32 (8H, m).

EXAMPLE 65 ##STR348## (1) EXAMPLE 65-1

Boc(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc (1)(7.7 g) was dissolved in water(150 ml) and the solution was adjusted to pH 8 with N-methylmorpholine.To the solution was added a dioxane solution (180 ml) ofD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)(oSu)(2)(containing 26.7 m mole ofcompound (2) as activated ester). The mixture was stirred for four hoursat ambient temperature. Dioxane was distilled off under reduced pressureand the resulting mixture was washed with ether (100 ml) and adjusted topH 3 with 5% aqueous hydrochloric acid. The solution was extracted withethyl acetate (300 ml and 150 ml) and the extract was washed with waterand dried over anhydrous magnesium sulfate and then evaporated todryness. The residue was crystallized from a mixture of chloroform andether (1:3) to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc(3)(10.8 g). mp 105°-113° C. (dec.).

N.M.R. (CD₃ OD), δ(ppm): 1.3-1.8 (30H, m), 2.08 (3H, s), 2.1-2.4 (4H,m), 3.92 (2H, s), 4.2-4.6 (4H, m), 4.8-5.1 (1H, m), 5.17 (2H, s), 7.33(5H, s)

(2) EXAMPLE 65-2

D-Lac(oAc)-L-Ala-γD-Glu(OH)oBzl (5.2 g) was dissolved in methylenechloride (60 ml) and N-methylmorpholine (1.10 g) was added thereto. Tothe mixture was added dropwise isobutyl chloroformate (1.50 g) at-10°--15° C. and the mixture was stirred at -10°--15° C. for thirtyminutes. To the mixture was added a solution prepared by dissolvingbis(trimethylsilyl)acetamide (9 ml) andBoc(D)meso-DAP(L)GlyOH-(D)-NHNHBoc (1)(4.60 g) in a mixture of methylenechloride (50 ml) and dimethylformamide (10 ml).

The resulting solution was stirred at -10°--15° C. for two hours and at0° C. for an hour and then concentrated to about 30 ml. To theconcentrate were added ethyl acetate (200 ml) and 2% aqueoushydrochloric acid (100 ml). The ethyl acetate layer was separated andwashed with water and then dried over anhydrous magnesium sulfate. Ethylacetate was evaporated to dryness under reduced pressure and the residuethus obtained was washed with ether to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc(3)(8.2 g) of which N.M.R. spectrum was identical with that of theproduct prepared in Example 65-1.

EXAMPLE 66 ##STR349##

ZDAP(OH)GlyOH (1)(80 mg) was added to a solution ofD-Lac(oAc)-L-Ala-γ-D-Glu(oSu)oBzl (2) (59 mg) in dimethylformamide (4 mlcontaining triethylamine (0.03 ml) at 0° C. The mixture was stirredovernight at ambient temperature and then evaporated to dryness. To theresidue thus obtained was added dil. hydrochloric acid and thenextraction was carried out with ethyl acetate. The extract was washedwith water and evaporated to dryness. The residue was recrystallizedfrom a mixture of hexane and chloroform to giveD-Lac-(OAC)-L-Ala-γ-D-Glu(α-oBzl)-Z-DAP(OH)GlyOH (3) (86 mg).

N.M.R. (CD₃ OD), δ(ppm): 1.35 (3H, d, J=7 Hz), 1.43 (3H, d, J=7 Hz),1.3-2.2 (8H, m), 2.12 (3H, s), 2.30 (2H, m), 3.95 (2H, s), 4.0-4.6 (4H,m), 5.12 (2H, s), 5.18 (2H, s), 7.36 (10H, s).

EXAMPLE 67 ##STR350##

Z-meso-DAPGlyOH (1) (1.23 g) and bis(trimethylsilyl)acetamide (4.00 g)were suspended in methylene chloride (50 ml) and the suspension wasstirred at ambient temperature for four hours. On the other hand,D-Lac(oAc)-L-Ala-γ-D-Glu(OH)oBzl (2) dicyclohexylamine salt (2.34 g) wasdissolved in methylene chloride (30 ml), and triethylamine hydrochloricacid salt (0.54 g) was added at ambient temperature to the solutionwhile stirring. The stirring was continued for two hours and thereaction mixture was cooled at -30° C. and a solution of isobutylchloroformate (0.53 g) in methylene chloride (50 ml) was added dropwisethereto. The resulting mixture was reacted for twenty minutes at thesame temperature. To the reaction mixture was added dropwise a methylenechloride solution of Z-meso-DAPGlyOH prepared above and the resultingmixture was stirred at -30° C. for an hour. The reaction mixture wasreacted at ambient temperature for four hours and filtered. The filtratewas washed with 10% aqueous hydrochloric acid and concentrated underreduced pressure and the residue thus obtained was extracted with ethylacetate. The ethyl acetate layer was washed with water and dried overanhydrous magnesium sulfate and concentrated under reduced pressure togive an oily D-Lac(oAc)-L-Ala-γ-D-Glu-(α-oBzl)-Z-meso-DAPGlyOH (3 g).

N.M.R. (CD₃ OD), δ(ppm): 1.35(3H,d,J=7 Hz), 1.43(3H,d,J=7 Hz),1.3-2.2(8H,m), 2112(3H,s), 2.30(2H,m), 3.95(2H,s), 4.0-4.6;l (4H,m),5.12(2H,s), 5.18(2H,s), 7.36(10H,s).

EXAMPLE 68 ##STR351##

To a mixture of D-Lac(oAc)-L-Ala-γ-D-Glu(OH)(α-oBzl) (1)(653 mg) andN-hydroxysuccinimide (178 mg) in dioxane (7 ml) was addedN,N'-dicyclohexylcarbodiimide (320 mg).

The mixture was stirred at 10° C. for ten minutes and further stirred atambient temperature for 14 hours. The reaction mixture was filtered andthe filtrate was evaporated to dryness and the residue was dissolved indioxane (4.5 ml). A 3 ml portion of this solution was added to a mixtureof Z-(L)-mesoDAP-(D)GlyOH(270 mg) and N-methylmorphorine (156 82 l) indioxane (4 ml) and the mixture was stirred at ambient temperature. After4.5 hours, a 0.9 mol portion of the above dioxane solution andN-methylmorphorine (100 μl) was added and the mixture was stirred at thesame temperature. After 4.5 hours an additional 0.6 ml of the abovedioxane solution was added and the mixture was stirred for 1.25 hours.The reaction mixture was washed with ether, acidified to pH 2 with adiluted aqueous hydrochloric acid and extracted with a mixture of ethylacetate and methylene chloride, containing 1N hydrochloric acid, waterand brine, dried over magnesium sulfate and evaporated to give anamorphous solid (660 mg) which was dissolved in chloroform andtriturated with ether to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(D)-Z-(L)-mesoDAP-(D)-GlyOH (3) (450mg).

N.M.R. (CDCl₃ -CD₃ OD), δ(ppm): 1.37 (3H, d, J=7 Hz), 1.45 (3H, d, J=7Hz), 1.2-1.9 (6H, m), 2.0-2.5 (4H, m), 3.93 (2H, broad s), 4.1-4.7 (3H,m), 5.10 (2H, s), 5.17 (2H, s).

EXAMPLE 69 ##STR352##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)meso-DAP(L)GlyOH-(D)-NHNHBoc(1)(9.4 g) was hydrogenated in acetic acid (100 ml) over 10% palladiumblack (2.0 g) for 2 hours under 1.5 hydrogen atmospheric pressure atambient temperature.

After completion of the reaction, the catalyst was filtered off and thefiltrate was evaporated to dryness under reduced pressure. The residuewas triturated with ether to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(L)GlyOH-(D)-NHNHBoc(2) (7.50 g).

N.M.R. (CD₃ OD), δ(ppm): 1.3-1.9 (m), 2.10 (3H, s), 2.1-2.4 (4H, m),3.90 (2H, s), 4.2-4.6 (4H, m), 4.8-5.1 (1H, m).

EXAMPLE 70 ##STR353##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)-Gly(OH)-(D)-NHNHBoc(1) (1.65g) was dissolved in 50% aqueous methanol (32 ml) and thesolution was stirred for 2.5 hours at ambient temperature, maintainingthe pH at 9.0 with 5% aqueous solution of potassium carbonate. Thesolution was evaporated to dryness under reduced pressure. The residuewas adjusted to pH 3 with 5% aqueous hydrochloric acid and then passedthrough a column packed with a macroporous non-ionic adsorption resin,HP20 (500 ml). The resin was washed with water (200 ml) and eluted witha mixture of methanol and water (1:1 volume). The eluate was evaporatedto dryness and the residue was washed with ether to giveD-Lac-L-Ala-γ-D-Glu(α--OH)-(L)-Boc-(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc (2)(1.24 g).

I.R.(Nujol): 3270, 3200-2600, 1720, 1640 cm⁻¹.

N.M.R. (CD₃ OD), δ(ppm): 1.47 (18H, s), 3.93 (2H, s).

EXAMPLE 71 ##STR354##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc(1) (1.0 g) was dissolved in 50% aqueous methanol (20 ml), and 1Naqueous sodium hydroxide (3.7 ml) was added thereto. The resultingsolution was stirred for two hours at ambient temperature andconcentrated to about 5 ml. The concentrate was adjusted to pH 3 andpassed through a column packed with a macroporous non-ionic adsorpotionresin, HP20 (24 ml). The column was washed with water (100 ml) andeluted with a mixture of methanol and water (1:1) and the eluate wasevaporated to dryness under reduced pressure. The residue thus obtainedwas washed with ether to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)GlyOH-(D)-NHNHBoc. (2)(660 mg) which was identified with the product prepared in Example 70.

EXAMPLE 72 ##STR355## (1) EXAMPLE 72-1

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc (1)(200 mg) was dissolved in trifluoracetic acid (1 ml) and the solutionwas stirred for fifteen minutes at ambient temperature. The solution wasevaporated to dryness under reduced pressure. The residue was trituratedwith ether. The powder thus obtained was dissolved in water (5 ml) and0.1N aqueous sulfuric acid (6.8 ml) was added. To the mixture was addeddropwise a solution of sodium periodate (146 mg) in water (2 ml). Themixture was stirred for an hour under ice-cooling and then the excessreagent was decomposed with sodium bisulfite. The resulting solution wasadjusted to pH 3 with an aqueous saturated sodium bicarbonate andconcentrated to 1 ml under reduced pressure. The concentrate was passedthrough a column packed with a macroporous non-ionic adsorption resin,HP20 and eluted with water. The eluate was lyophilized to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP(L)GlyOH (2) (86 mg) as a solid.

N.M.R. (D₂ O), δ(ppm): 1.40 (3H, d, J=7 Hz), 1.46 (3H, d, J=7 Hz), 3.88(1H, t, J=5 Hz), 4.02 (2H, s).

[α]_(D) =-30.0 (c=0.4, water).

(2) EXAMPLE 72-2

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc (1)(220 mg) was dissolved in trifluoroacetic acid (1.5 ml) and the solutionwas stirred for fifteen minutes at ambient temperature. Thetrifluoroacetic acid was evaporated to dryness under reduced pressureand the residue thus obtained was triturated with ether. The powder wasdissolved in a mixture of water (5 ml) and dioxane (7 ml), andN-bromosuccinimide (110 mg) was added under ice-cooling thereto and thesolution was stirred for an hour. The reaction mixture was concentratedto about 1 ml and the concentrate was adjusted to pH 2.5 with 5% aqueoussodium bicarbonate. The solution was passed through a column packed witha macroporous non-ionic adsorption resin, HP20 (16 ml) and elution wascarried out with water and the eluate was lyophilized to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP(L)GlyOH (2) (150 mg), which wasidentified with the product prepared in Example 72-1.

(3) EXAMPLE 72-3

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc (1)(200 mg) was dissolved in trifluoroacetic acid (1 ml) and the solutionwas stirred at ambient temperature for fifteen minutes. Trifluoroaceticacid was evaporated to dryness under reduced pressure. The residue thusobtained was triturated and the powder was dissolved in 60% aqueousacetic acid (15 ml). To the solution was added manganese dioxide (65 mg)while stirring at ambient temperature and the mixture was stirred at thesame temperature for 1.5 hours. Insoluble materials were filtered offand then the filtrate was evaporated to dryness under reduced pressure.The residue thus obtained was dissolved in 50% aqueous ethanol andcooled to -10°--20° C. and then adjusted to pH 9 with aqueous ammonium.The solution was allowed to stand at the same temperature for two hours.Insoluble materials were filtered off and the filtrate was evaporated todryness under reduced pressure. The residue was passed through a columnpacked with activated carbon (10 ml). The column was washed with water(30 ml) and eluted with 70% aqueous acetone. The fraction containing theobject compound was collected and evaporated to dryness under reducedpressure. The residue was pulverized with a mixture of methanol andether to give D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP(L)GlyOH (2) (84mg), whose structure was confirmed in comparison of data of the productsprepared in Example 72-1.

EXAMPLE 73 ##STR356##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-ZDAP(OH)GlyOH (1) (2.80 g) wasdehydrogenated in acetic acid (15 ml) over 10% palladium black (0.50 g)under two atmospheric pressure of hydrogen for six hours. The reactionmixture was filtered and the filtrate was concentrated to give an oil.The oil was dissolved in water and the solution was adjusted to pH9.0-9.5 with diluted aqueous solution of potassium carbonate, and thenstirred under ice-cooling for four hours. The reaction mixture wasadjusted to pH 7 with dil. hydrochloric acid and concentrated. Theconcentrate was dissolved in methanol and insoluble materials werefiltered off. The methanolic filtrate was evaporated to dryness underreduced pressure, and the residue was dissolved in water (3 ml) adjustedto pH 3-3.2, pass through a column packed with a macroporous non-ionicadsorption resin, HP20 (200 ml) and then eluted with water. Thefractions containing the object compound were collected and concentratedto give a powder (400 mg). The powder was dissolved in water and thesolution was again passed through a column packed with a macroporousnon-ionic adsorption resin, HP20 (100 ml). Elution was carried out withwater and the fraction containing the object compound was collected andconcentrated to give D-Lac-L-Ala-γ-D-Glu(α-OH)-DAP(OH)GlyOH (2) (100mg), whose structure was confirmed in comparison of data of the productsprepared in Example 72-1.

EXAMPLE 74 ##STR357##

5% Aqueous potassium bicarbonate (3.25 ml) was added to a solution ofD-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(D)-mesoDAP(D)GlyOH (1) (278 mg) in 5%aqueous methanol (6 ml) at ambient temperature. The mixture was stirredat ambient temperature with occasional addition of 5% aqueous potassiumbicarbonate in order to maintain pH 9. The reaction mixture wasacidified to pH 3.5 with 1N aqueous hydrochloric acid and evaporated todryness. The residue was dissolved in water and the solution wasconcentrated to about 2 ml. The concentrate was chromatographed onmacroporous non-ionic adsorption resin, HP20 (15 ml) and elution wascarried out with water. The fraction containing the object compound wasevaporated, and the residue was dissolved in a small amount of water andlyophilized to give D-Lac-L-Ala-γ-D-Glu(α-OH)-(D)-mesoDAP-(D)-GlyOH (135mg), whose structure was confirmed in comparison of data of the productsprepared in Example 72-1.

EXAMPLE 75 ##STR358##

A mixture ofD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(D)-Z-(L)-mesoDAP-(D)-GlyOH (1) (440mg) and 10% palladium black (90 mg) in acetic acid (5 ml) was stirredunder hydrogen atmosphere for four hours. Additional 10% palladium black(60 mg) and acetic acid (3 ml) were added to the reaction mixture andthe stirring was continued under hydrogen atmosphere for four hours. Thereaction mixture was filtered. The filtrate was concentrated underreduced pressure and the residue was dissolved in water. The aqueoussolution was evaporated to dryness. The residue was again dissolved inwater and concentrated. The concentrate was lyophilized to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(D)-meso-DAP(D)GlyOH (2) (301 mg).

N.M.R. (D₂ O), δ(ppm): 1.42 (3H, d, J=7 Hz), 1.47 (3H, d, J=7 Hz), 2.14(3H, s), 1.0-2.8 (10H, m), 3.80 (1H, t, J=7 Hz), 3.94 (2H, s), 4.1-4.6(3H, m) 5.02 (1H, q, J=7 Hz).

EXAMPLE 76 ##STR359##

A solution of D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-Z-DAP(OH)GlyOH inmethanol (1) (20 mg) was hydrogenated over 10% palladium black (50 mg).The reaction mixture was filtered and the filtrate was evaporated todryness to give D-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-DAP(OH)GlyOH (2) (10 mg),whose structure was confirmed in comparison of data of the productsprepared in Example 75.

EXAMPLE 77 ##STR360##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-DAP(OH)GlyOH (1) (10 mg) was added to0.05M solution of potassium carbonate in 70% methanol (0.6 ml) at 0° C.and the mixture was stirred at ambient temperature for an hour. Thereaction mixture was neutralized with acetic acid and then evaporated togive a residue. The residue was purified by suing carbon columnchromatography. Elution was carried out with 50% aqueous acetone to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-DAP(OH)GlyOH (2) (7 mg), whose structure wasconfirmed in comparison of data of the products prepared in Example72-1.

EXAMPLE 78 ##STR361##

To a mixture of D-Lac(oAc)-L-Ala-D-Glu(OH)oBzl (1) (2.45 g) and succinicacid (740 mg) in dioxane (20 ml) was added N,N'-dicyclohexylcarbodimido(1.25 g) at 12° C. and the mixture was stirred at ambient temperaturefor 16 hours. The reaction mixture was filtered andN,N'-dicyclohexylurea was washed with dioxane.

The filtrate and the washings were combined and concentrated. Theconcentrate was dissolved in dioxane (18 ml). This solution was used forthe next reaction. To a mixture of Z-(α)-L-DAP-(ε)-GlyOH (2) (1.21 g)and N-methylmorpholine (1.02 ml) in water (20 ml) was added the abovesolution at 5° C. and the mixture was stirred at 5° C. for 40 minutesand at ambient temperature for 4 hours. An additional N-methylmorpholine(0.2 ml) was added to the reaction mixture and the resulting mixture wasstirred at the same temperature for two hours. The reaction mixture wasconcentrated and the concentrate was diluted with water. The solutionwas washed with ether and acidified with dil. hydrochloric acid to pH 2.To the solution was added a mixture of methylene chloride and ethylacetic and the mixture was shaked and then filtered to give insolublematerials and the organic layer. The organic layer was washed withwater, dried over magnesium sulfate and concentrated. The residue wascombined with the soluble materials described above and dissolved in amixture of chloroform and methanol (1:1). The solution was concentratedand the concentrate was triturated with a mixture of ethyl acetate andether (1:2) to give a powder (1.8 g), which was dissolved in a mixtureof chloroform and methanol and ethyl acetate (1:1:1) and concentrated.The concentrate was triturated with a mixture of ethyl acetate and ether(5:1) to give D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(α)-Z-(ε)-L-DAP-(α)-GlyOH(3)(1.654 g).

N.M.R. (CDCl₃ -CD₃ OD), δ(ppm): 1.37 (3H, d, J=7 Hz), 1.46 (3H, d, J=7Hz). 2.11 (3H, s), 1.2-2.6 (10H, m), 3.95 (2H, s), 4.0-4.6 (4H, m), 4.96(1H, q, J=7 Hz), 5.08 (2H, s), 5.15 (2H, s), 7.33 (10H, s).

EXAMPLE 79 ##STR362##

A solution of D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(α)-Z-(ε)-L-DAP-(α)-GlyOH(1)(1.59 g) in acetic acid (40 ml) was hydrogenated over 10% palladiumblack (500 mg) for 4 hours. The reaction mixture was filtered and thefilter cake was washed with 50% aqueous methanol. The filtrate and thewashings were combined and evaporated and pumped to give a residue (1.5g). Contaminated acetic acid was removed by co-eveporation with waterand this operation was repeated once more. The residue was dissolved inwater and lyophilized to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(α)-L-DAP-(α)-GlyOH (2)(1.10 g).

N.M.R. (D₂ O), δ(ppm): 1.43 (3H, d, J=7 Hz), 1.48 (3H, d, J=7 Hz),1.3-2.7 (10H, m), 2.15 (3H, s), 3.80 (1H, broad t, J=6 Hz), 3.95 (2H,s), 4.0-4.6 (3H, m), 5.05 (1H, q, J=7 Hz).

EXAMPLE 80 ##STR363##

To a solution of D-Lac-(oAc)-L-Ala-γ-D-Glu(α-OH)-(α)-L-DAP-(α)-GlyOH (1)in a mixture of methanol (15 ml) and water (5 ml) was added 1N sodiumhydroxide (7.5 ml) at 5° C. The solution was stirred at the sametemperature for 30 minutes and at ambient temperature for 2.25 hours.

The resulting solution was acidified to pH 4 with 3N hydrochloric acidand concentrated. The concentrate was diluted with water to 10 ml,acidified to pH 2.2 with 3N hydrochloric acid and chromatographed on acolumn of a macroporous non-ionic adsorption resin, HP 20 (135 ml).Elution was carried out with water and the eluate was concentrated andlyophilized to give D-Lac-L-Ala-γ-D-Glu(α-OH)-(α)-L-DAP-(α)-GlyOH (2)(681 mg). mp. 130° C. (dec.)

N.M.R. (D₂ O), δ(ppm): 1.39 (3H, d, J=7 Hz), 1.45 (3H, d, J=7 Hz),1.1-2.6 (10H, m), 3.86 (1H, t, J×6 Hz), 4.00 (2H, s), 4.1-4.6 (4H, m).

[α]_(D) ¹⁵ =-23.3° (C=0.322, water).

EXAMPLE 81

(1) Step 1 ##STR364##

D-Lac(OAc)-L-Ala-γ-L-Glu(α-OBzl)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc(3) was prepared from Boc(D)-meso-DAP-(L)GlyOH-(D)-NHNHBoc (1) andD-Lac(OAc)-L-Ala-L-Glu(α-OBzl)(Z) in substantially the same manner asthat of Example 65-1.

IR(Nujol): 3300, 1735, 1695 1645 cm⁻¹.

NMR(CD₃ OD): 1.40(9H, S), 1.42(9H, S), 2.08(3H, S), 3.90(2H, broad S),4.00-5.00(5H, m), 5.10(2H, S), 7.30(5H, S).

(2) Step 2 ##STR365##

D-Lac(OH)-L-Ala-γ-L-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(L)-GlyOH-(D)-NHNHBoc(4) was prepared in substantially the same manner as that of Example 71.

NMR(CD₃ OD), δ(ppm): 1.44(9H, S), 1.46(9H, S), 3.90(2H, broad S),4.00-4.60(5H, m).

(3) Step 3 ##STR366##

D-Lac(OH)-L-Ala-γ-L-Glu(α-OH)-(L)-meso-DAP(L)GlyOH (5) was prepared insubstantially the same manner as that of Example 72-1.

NMR(D₂ O), δ(ppm): 1.38(3H,d, J=7 Hz), 1.00-2.60(10H, m), 3.98(2H, S),3.84(1H, t, J=7 Hz), 4.1-4.5(4H, m).

[α]_(D) =-34.7 (c=0.15 water).

EXAMPLE 82

(1) Step 1 ##STR367##

L-Lac(OAC)-L-Ala-γ-D-Gln(α-OBzl)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc(3) was prepared from Boc (D)-mesoDAP-(L)-GlyOH-(D)-NHNHBoc (1) andL-Lac(OAC)-L-Ala-D-Glu(α-OBzl) (2) in substantially the same manner asthat of Example 65-1.

(2) Step 2 ##STR368##

L-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc(4) was prepared in substantially the same manner as that of Example 71.

Step (3) ##STR369##

L-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP(L)GlyOH (5) was prepared insubstantially the same manner as that of Example 72-1.

mp 170°-174° C. (dec).

[α]_(D) =-33.2° (C=0.25, water)

NMR (D₂ O), δ(ppm): 1.38(3H, ,J=7 Hz), 1.45(3H, ,J=7 Hz), 3.89(1H,t,J=7Hz), 4.01(2H,s), 4.20 4.55(m).

EXAMPLE 83 (1) Step 1 ##STR370##

D-Lac(OAC)-D-Ala-γ-L-Glu(α-OBzl)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc(3) was prepared from Boc(D)-mesoDAP-(L)-GlyOH-(D)-NHNHBoc (1) andD-Lac(OAC)-D-Ala-L-Glu(α-OBzl) (2) in substantially the same manner asthat of Example 65-1.

NMR (CD₃ OD), δ(ppm): 1.40(9H, s), 1.42(9H,s), 2.06(3H,s), 3.90(2H,broads), 3.90-5.00(5H,m), 5.15(2H,s), 7.35(5H,s).

(2) Step 2 ##STR371##

D-Lac(OH)-D-Ala-γ-L-Glu(α-OH)-(L)-Boc-(D)-meso-DAP(L)-GlyOH-(D)-NHNHBoc(4) was prepared in substantially the same manner as that of Example 71.

IR (Nujol): 3300, 1740, 1690 1740 (Broad) cm⁻¹.

NMR (CD₃ OD), δ(ppm): 3.90(2H,broad s), 4.00-4.60(5H,m).

(3) Step 3 ##STR372##

D-Lac-(OH)-D-Ala-γ-L-Glu(α-OH)-(L)-mesoDAP(L)GlyOH (5) was prepared insubstantially the same manner as that of Example 72-1.

NMR (D₂ O), δ(ppm): 1.35(3H,d,J=7 Hz), 1.40(3H,d,J=7 Hz),1.00-2.60(10H,m), 3.80(1H,t,J=7 Hz), 3.90(2H,s), 4.10-4.5(4H,m).

[α]_(D) =+0.76 (c=0.35, water).

EXAMPLE 84

(1) Step 1 ##STR373##

D-Lac(OAC)-D-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) was prepared from Boc(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (1) andD-Lac(oAC)-D-Ala-D-Glu(α-OBzl) (2) in substantially the same manner asthat of Example 65-1.

N.M.R. (CD₃ OD), δ(ppm): 1.41 (18H, s), 2.12 (3H, s), 3.92 (2H, broads), 4.00-5.10 (5H, m), 5.16 (2H, s), 7.36 (5H, s).

(2) Step 2 ##STR374##

D-Lac(OH)-D-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(4) was prepared in substantially the same manner as that of Example 71.

N.M.R. (CD₃ OD), δ(ppm): 1.41 (18H, s), 3.92 (2H, broad s), 4.00-4.60(5H, m)

(3) Step 3 ##STR375##

D-Lac (OH)-D-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (5) was prepared insubstantially the same manner as that of Example 72-1.

[α]_(D) =11.6 (c=0.25, water).

N.M.R. (CD₃ OD), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.44 (3H, d, J=7 Hz),1.00-2.60 (10H, m), 3.88 (1H, t, J=7 Hz), 3.98 (2H, broad s), 4.10-4.50(5H, m).

The following compounds were prepared in substantially the same manneras that of Steps 1 and 2 of Example 1.

EXAMPLE 85

(1) Step 1

Benzenesulfonyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH.

NMR (CD₃ OD), δ(ppm): 1.24 (3H, d, J=7 Hz), 1.47 (18H, s), 1.25-2.46(10H, m), 3.94 (2H, s), 3.82-4.54 (4H, m), 7.55-8.00 (5H, m).

(2) Step 2

Benzenesulfonyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH.

NMR (D₂ O), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.5-2.42 (10H, m), 3.67-4.42(4H, m), 4.10 (2H, s), 7.54-7.97 (5H, m).

EXAMPLE 86

(1) Step 1

Cinnamoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOHwas prepared in substantially the same manner as that of Step 1 ofExample 1.

NMR (CD₃ OD), δ(ppm): 1.34 (13H, m), 1.44 (18H, s), 3.95 (2H, s),3.90-4.67 (4H, m), 6.74 (1H, d, J=7 Hz), 7.57 (1H, d, J=16 Hz),7.32-7.70 (5H, m).

(2) Step 2

Cinnamoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-NHNH₂ -(L)-GlyOH wasprepared in substantially the same manner as that of Step 3 of Example11.

NMR (D₂ O), δ(ppm): 1.47 (3H, d, J=7 Hz), 1.34-2.42 (10H, m), 3.95 (2H,s), 3.78-4.58 (4H, m), 6.64 (1H, d, J=16 Hz), 7.37-7.70 (6H, m).

(3) Step 3

Cinnamoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH was prepared insubstantially the same manner as that of Step 4 of Example 11.

NMR (D₂ O), δ(ppm): 1.46 (3H, d, J=7 Hz), 1.28-2.44 (10H, m), 3.72 (1H,t, J=7 Hz), 3.88 (2H, s), 4.20-4.46 (4H, m), 6.67 (1H, d, J=7 Hz),7.38-7.46 (6H, m).

EXAMPLE 87

(1) Step 1 ##STR376##

To a mixture of D-Lac(OAc)-L-Ala-D-GluOBzl (2) (1.20 g) andN-methylmorpholine (0.28 ml) in methylene chloride (17 ml) was addedisobutyl chlorocarbonate (0.33 ml). The resulting mixture was stirred at-15° C. to -10° C. for 30 minutes and then cooled to -40° C. To theresulting mixture was added a mixture of Z-D-LysOH (1) (725 ml) andbis(trimethylsilyl)acetamide (3 ml) in dimethylformamide (3 ml) andmethylene chloride (8 ml) at -40° C. The resulting mixture was stirredat -15° C. to -10° C. for an hour and gradually allowed to warm toambient temperature. The resulting mixture was concentrated, taken upinto ethyl acetate (80 ml) and washed with dil hydrochloric acid, waterand brine, in turn. The concentrate was dried over magnesium sulfate andthe solvent was distilled off to give an oil which was crystallized froma mixture of chloroform and isopropyl ether to giveD-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-ε-Z-D-LysOH (3)(1.60 g).

NMR (DMSO-d₆), δ(ppm): 1.28 (3H, d, J=7 Hz), 1.37 (3H, d, J=7 Hz),1.0-2.4 (10H, m), 2.10 (3H, s), 3.1 (2H, m), 4.0 (1H, m), 4.40 (2H,broad t, J=7 Hz), 5.05 (1H, q, J=6.5 Hz), 5.10 (2H, s), 5.20 (2H, s),7.42 (10H, s,) 7.3-8.0 (2H, m), 8.0-8.4 (2H, m).

(2) Step 2 ##STR377##

A solution of D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-ε-Z-D-LysOH (3)(1.50 g)in acetic acid (40 ml) was hydrogenated over 10% palladium black (500mg) at ambient temperature under an atmospheric pressure of hydrogen.The reaction mixture was filtered and the catalyst was washed with 50%aqueous methanol. The filtrates were combined and evaporated. Aceticacid was removed by co-evaporation with water. The residue was dissolvedin water and lyophilized to giveD-Lac(OAc)-L-Ala-γ-D-Glu(α-OH)-ε-D-LysOH (4)(1.00 g).

NMR (D₂ O), δ(ppm): 1.42 (3H, d, J=7 Hz), 1.47 (3H, d, J=7 Hz), 2.16(3H, s), 1.2-2.5 (10H, m). 3.0-3.4 (2H, m), 3.83 (1H, t, J=6 Hz),4.1-4.6 (2H, m), 5.07 (1H, q, J=7 Hz).

(3) Step 3 ##STR378##

To a solution of D-Lac(OAc)-L-Ala-γ-D-Glu(α-OH)-ε-D-LysOH (4)(930 mg) ina mixture of methanol (9 ml) and water (3 ml) was added dropwise 1Nsodium hydroxide (6 ml). The resulting solution was stirred at 5° C. foran hour and at ambient temperature of two hours.

The reaction mixture was neutralized to pH 7 with 1N hydrochloric acidand concentrated. The concentrate was adjusted to pH 2.2 with 1Nhydrochloric acid and chromatographed on a macroporous non-ionicadsorption resin, HP 20 (90 ml), eluting with water. Fractionscontaining the object compound (5) were collected, concentrated andlyophilized to give D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-ε-D-LysOH (5)(700 mg).

[α]_(D) =-17.1° C. (C=0.509, water).

NMR (D₂ O), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.42 (3H, d, J=7 Hz), 1.1-2.6(10H, m), 3.19 (2H, broad t, J=7 Hz), 3.83 (1H, t, J=6 Hz), 4.1-4.5 (3H,m).

EXAMPLE 88

(1) Step 1 ##STR379##

D-Lac(OAc)-L-Ala-β-D-Asp(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) was prepared from D-Lac(OAc)-L-Ala-D-Asp(OH)OBzl (1) andBoc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (2) in substantially the samemanner as that of Example 66.

m.p. 120°-123° C. (dec.).

I.R. (Nujol): 3550, 3300, 1730, 1650, 1550 cm⁻¹.

NMR (DMSO-d₆), δ(ppm): 1.0-1.9 (30H, m), 2.06 (3H, s), 3.0-5.0 (13H, m),3.18 (2H, s), 5.14 (2H, s), 6.63 (1H, m), 7.40 (5H, s), 7.9-8.6 (6H, m),9.56 (1H, s).

(2) Step 2 ##STR380##

To a suspension ofD-Lac(OAc)-L-Ala-β-D-Asp(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) (0.74 g) in water (10 ml) was added 1N sodium hydroxide (3.0 ml) at0° C. and the mixture was stirred for 1 hour at the same temperature.The reaction mixture was neutralized to pH 3 with 1N hydrochloric acid(3.0 ml) and evaporated to give an oil, which was dissolved in water (3ml) and applied to a column of HP-20 (20 ml). The column was washed withwater and eluted with methanol:water (7:3) and the eluate was evaporatedto give an oil (0.42 g), which was then dissolved in trifluoracetic acid(5 ml) and stirred for 15 minutes at room temperature. After evaporationof trifluoroacetic acid, the resulting residue was pulverized with etherand filtered to give a powder, which was dissolved in 1N sulfuric acid(2.5 ml) and sodium periodate (0.09 g) was added at 0° C. After stirringfor 10 minutes at the same temperature, the reaction mixture was treatedwith 0.5N aqueous sodium hydrogen sulfite (0.2 ml). This solution wasneutralized to pH 2 with 1M sodium carbonate (1.4 ml) and evaporated andmethanol (3 ml) was added and an undissolved material was filtered off.The filtrate was evaporated to give an oil (0.30 g), which was dissolvedin a small amount of water and applied to a column of HP-20 (40 ml). Thecolumn was eluted with water and the eluate was evaporated to give anoil (140 mg), which was again applied to a column of HP-20 (60 ml) andeluted with water. The eluate was evaporated and the residue wasdissolved in a small amount of water and lyophilized to giveD-Lac-L-Ala-β-D-Asp(α-OH)-(L)-mesoDAP-(L)-GlyOH (4) (120 mg) as a whitepowder.

I.R. (Nujol): 3270, 1720, 1640, 1520 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.38 (3H, J=7 Hz), 1.2-2.1(6H, m), 2.6-3.0 (2H, m), 3.87 (1H, t, J=6 Hz), 3.95 (2H, s), 4.1-4.5(4H, m).

EXAMPLE 89

(1) Step 1 ##STR381##

To a mixture of Boc-L-Ala-D-Glu(OH)OMe (1) (1.94 g), L-Lys(ε-Z)-GlyOEt(2)(1.67 g) and 1-(4-chlorobenzenesulfonyloxy)-6-chlorobenzotriazole(1.72 g) in methylene chloride (100 ml) was added N-methylmorpholine(1.01 g) and the mixture was stirred for 2 days at room temperature. Thereaction mixture was washed sccessively with 5% aqueous sodiumbicarbonate, water, 5% hydrochloric acid and water and dried overmagnesium sulfate. After evaporation of the solvent, the resultingcrystalline mass was filtered and washed with ether, to giveBoc-L-Ala-γ-D-Glu(α-OMe)-L-Lys(ε-Z)-GlyOEt (3)(1.34 g).

m.p. 126°-127° C.

I.R. (Nujol): 3550, 3255, 1750, 1720, 1700, 1680, 1660, 1650, 1640 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 1.22 (3H, t, J=7 Hz), 1.30 (3H, d, J=7 Hz), 1.42(9H, s), 1.34-1.60 (6H, m), 2.70 (2H, t, J=7 Hz), 3.08 (2H, m), 3.68(3H, s), 3.92 (2H, s), 4.15 (2H, q, J=7 Hz), 4.00-4.42 (3H, m), 5.07(2H, s), 7.34 (5H, s).

(2) Step 2 ##STR382##

To a solution of Boc-L-Ala-γ-D-Glu(α-OMe)-L-Lys(ε-Z)-GlyOEt (3)(1.20 g)in a mixture of ethyl acetate (20 ml) and methanol (5ml) was added asaturated solution of hydrogen chloride in ethyl acetate (120 ml) at 0°C. and the mixture was stirred for 20 minutes at the same temperatureand for 30 minutes at room temperature. The reaction mixture wasevaporated to give a powder, which was washed successively with ethylacetate and ether to give L-Ala-γ-D-Glu(α-OMe)-L-Lys(ε-Z)-GlyOEt.HCl(4)(0.97 g).

NMR (D₂ O), δ(ppm): 1.25 (3H, t, J=7 Hz), 1.62 (3H, t, J=7 Hz),1.34-1.84 (8H, m), 2.44 (2H, t, J=6 Hz), 3.17 (2H, t, J=6 Hz), 3.82 (3H,s), 4.04 (2H, s), 4.12 (2H, q, J=7 Hz), 4.17-4.68 (3H, m), 5.12 (2H, s),7.42 (5H, s).

(3) Step 3 ##STR383##

To a mixture of L-Ala-γ-D-Glu(α-OMe)-L-Lys(ε-Z)-GlyOEt.HCl (4)(0.90 g)and triethylamine (0.17 g) in methylene chloride (15 ml) was added asolution of acetyl D-lactyl chloride (0.26 g) in methylene chloride (5ml) at 5° C. and the mixture was stirred for 1 hour at the sametemperature. The reaction mixture was washed successively with 5%aqueous sodium bicarbonate, water, 5% hydrochloric acid and water, driedover magnesium sulfate and evaporated to give a crystalline mass, whichwas washed with ethyl acetate to giveD-Lac(OAc)-L-ALa-γ-D-Glu(α-OMe)-L-Lys(ε-Z)-GlyOEt (5)(0.97 g).

I.R. (Nujol): 3400, 1730-1640 (broad) cm⁻¹.

NMR (D₂ O), δ(ppm): 1.17 (3H, t, J=7 Hz), 1.45 (3H, d, J=7 Hz),1.34-1.67 (8H, m), 2.04 (3H, s), 1.96-2.13 (2H, m), 3.07-2.87 (2H, m),3.64 (3H, s), 4.07 (2H, d, J=7 Hz), 4.25 (2 H, q, J=7 Hz), 3.57-4.34(3H, m), 5.00 (2H, s), 7.34 (5H, s), 8.25-7.84 (4H, m).

(4) Step 4 ##STR384##

To a solution of D-Lac(OAc)-L-Ala-γ-D-Glu-L-Lys(ε-Z)-GlyOEt (5)(0.90 g)in methanol (15 ml) was added 1N sodium hydroxide (30 ml) at 5° C. andthe mixture was stirred for 4 hours at the same temperature andovernight at room temperature. Methanol was evaporated and the resultingaqueous layer was adjusted to pH 5.5 with diluted hydrochloric acid andwashed with ethyl acetate. The aqueous layer was acidified to pH 1 withdiluted hydrochloric acid and extracted with ethyl acetate. The extractwas washed with water saturated with sodium chloride, dried overmagnesium sulfate and evaporated to give an amorphous solid (0.50 g).This solid was dissolved in MeOH (15 ml) and hydrogenated over 5%palladium-charcoal (0.10 g). After removal of the catalyst byfiltration, the filtrate was evaporated and the resulting residue waspulverized with acetone to give D-Lac-L-Ala-γ-D-Glu(α-OH)-L-Lys-GlyOH(0.25 g) as a powder.

I.R. (Nujol): 3350, 1710, 1670-1640 cm⁻¹.

NMR (D₂ O+NaHCO₃), δ(ppm): 1.37 (3H, d, J=7 Hz). 1.44 (3H, d, J=7 Hz),1.34-2.00 (8H, m). 1.84-2.25 (2H, m), 3.04 (2H, t, J=7 Hz). 3.79 (2H,s), 4.17-4.50 (3H, m).

EXAMPLE 90

(1) Step 1 ##STR385##

To a mixture of Boc-L-Ala-D-Glu(OH)OMe (1)(1.33 g), L-Lys(ε-Boc)-GlyOEt(2)(2.02 g) and 1-(4-chlorobenzenesulfonyloxy)-6-chlorobenzotriazole(1.38 g) in methylene chloride (50 ml) was added N-methylmorphorine(0.808 g) at 0° C. and the mixture was stirred for 30 minutes at thesame temperature and for 2 days at room temperature. The reactionmixture was washed successively with 5% aqueous sodium bicarbonate,water, 5% hydrochloride acid and water, dried over magnesium sulfate andevaporated to give a crystalline mass, which was washed with ether togive Boc-L-Ala-γ-D-Glu(α-OMe)-L-Lys(ε-Boc)-GlyOEt (3)(1.96 g).

m.p. 163°-4° C.

I.R. (Nujol): 3550, 3275, 1740, 1690, 1650 cm⁻¹.

NMR (DMSO-d₆), δ(ppm): 1.17 (3H, t, J=7 Hz), 1.32 (3H, d, J=7 Hz), 1.35(18H, s), 1.42-1.54 (8H, m), 2.08-2.18 (2H, m), 3.25-2.82 (2H, m), 3.62(3H, s), 3.80 (2H, d, J=6 Hz), 4.07 (2H, q, J=7 Hz), 3.80-4.07 (3H, m),6.58-6.77 (2H, m), 7.83-8.25 (3H, m).

(2) Step 2 ##STR386##

To a solution of Boc-L-Ala-γ-D-Glu(α-OMe)-L-Lys(ε-Boc)-GlyOEt (3)(0.50g) in methanol (5 ml) was added 1N sodium hydroxide (2 ml) at 0° C. andthe mixture was stirred for 1 hour at the same temperature and overnightat room temperature. The reaction mixture was evaporated and the residuewas dissolved in water and washed with ethyl acetate. The aqueous layerwas acidified with diluted hydrochloric acid to pH 2 and extracted withethyl acetate. The extract was washed with water, dried over magnesiumsulfate and evaporated to give an oil (0.37 g). This oil was dissolvedin ethyl acetate (10 ml) and a saturated solution of hydrogen chloridein ethyl acetate (5 ml) was added at 0° C. The mixture was stirred for10 minutes at the same temperature and for 30 minutes at roomtemperature. The resulting precipitate was filtered and washed withethyl acetate to give L-Ala-γ-D-Glu(α-OH)-L-Lys-GlyOH (4)(0.17 g) as apowder.

I.R. (Nujol): 3450, 3200, 1730, 1680, 1660-1640 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.65 (3H, d, J=7 Hz), 1.92-1.42 (8H, m), 2.34 (2H,t, J=6 Hz), 2.64 (2H, t, J=6 Hz), 3.95 (2H, s), 4.10-4.67 (3H, m).

EXAMPLE 91

(1) Step 1 ##STR387##

D-Lac(OAc)-L-Ala-D-Aad(OH)oBzl(3) was prepared from O-acetyl-D-lactylchloride (1) and L-Ala-D-GluOBzl (2) in substantially the same manner asthat of Step 1 of Example 1.

I.R. (CHCl₃): 3400, 3300, 1730, 1710, 1665 cm⁻¹.

NMR (CDCl₃), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.41 (3H, d, J=7 Hz), 1.4-2.0(4H, m), 2.08 (3H, s), 2.2-2.4 (2H, m), 4.4-4.7 (2H, m), 4.9-5.2 (1H,m), 5.10 (2H, s), 6.96 (1H, d, J=8 Hz), 7.15 (1H, d, J=8 Hz), 7.20 (5H,s), 7.4-8.2 (1H, broad s).

(2) Step 2 ##STR388##

D-Lac(OAc)-L-Ala-δ-D-Aad(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(5) was prepared from D-Lac(OAc)-L-Ala-D-Aad(OH)oBzl(3) andBoc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (4) is substantially the samemanner as that of Example 66.

m.p. 108°-110° C.

I.R. (Nujol): 3260, 1725, 1670, 1650, 1630, 1540, 1520 cm⁻¹.

NMR (CDCl₃), δ(ppm): 1.40 (3H, d, J=7 Hz), 1.4-2.0 (13H, m), 1.44 (18H,s), 2.10 (3H, s), 2.1-2.5 (2H, m), 3.90 (2H, s), 3.8-4.2 (1H, m),4.3-4.6 (3H, m), 4.7-5.1 (1H, m), 5.12 (2H, s), 7.30 (5H, s).

(3) Step 3 ##STR389##

D-Lac-L-Ala-δ-DAad(α-OH)-(L)-mesoDAP-(L)-GlyOH (6) was prepared fromD-Lac(OAc)-L-Ala-δ-D-Aad-(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(5) in substantially the same manner as that of Step 2 of Example 88.

I.R. (Nujol): 3270, 1720, 1620, 1530 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.43 (3H, d, J=7 Hz), 1.5-2.1(10H, m), 2.2-2.5 (2H, m), 3.84 (1H, t, J=6 Hz), 3.96 (2H, s), 4.1-4.5(4H, m).

EXAMPLE 92

(1) Step 1 ##STR390##

D-Lac(OAc)-L-Ala-γ-Abu-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH (3) wasprepared in substantially the same manner as that of Example 66.

NMR (CDCl₃), δ(ppm): 1.2-2.0 (4H, m), 1.42 (18H, s), 2.0-2.5 (2H, m),2.15 (3H, s), 3.1-3.4 (2H, m), 3.8-4.1 (2H, m), 4.3-4.7 (1H, m), 4.9-5.2(1H, m), 5.5-6.0 (2H, m), 7.3-7.6 (2H, m), 7.6-8.1 (2H, m).

(2) Step 2 ##STR391##

D-Lac(OH)-L-Ala-γ-Abu-(L)-mesoDAP-(L)-GlyOH (4) was prepared insubstantially the same manner as that of Example 88.

NMR (D₂ O), δ(ppm): 1.39 (3H, d, J=7 Hz), 1.40 (3H, d, J=7 Hz), 1.5-2.1(8H, m), 2.33 (2H, t, J=7 Hz), 3.21 (2H, t, J=7 Hz), 3.79 (1H, t, J=7Hz), 3.95 (2H, s), 4.1-4.5 (4H, m).

The following compounds were prepared in substantially the same manneras Steps 1 and 2 of Example 1, respectively.

EXAMPLE 93

(1) Step 1

Thienylacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc.

NMR (DMSO-d₆), δ(ppm): 1.36 (18H, s), 1.00-2.33 (10H, m), 3.66 (2H, s),3.83-4.33 (3H, m), 6.87 (2H, m), 7.27 (1H, m), 7.83-8.66 (3H, m), 9.02(2H, broad s).

(2) Step 2

Thienylacetyl-γ-D-Glu(α-(OH)-(L)-mesoDAP.

NMR (D₂ O), δ(ppm): 1.20-2.60 (10H, m), 3.78 (1H, t, J=7 Hz), 3.86 (2H,s), 4.28 (1H, t, J=7 Hz), 7.04 (2H, m), 7.32 (2H, m).

EXAMPLE 94: For fermentation

(1) A vegetative medium 1 (pH 7.0) was prepared from the followingingredients:

    ______________________________________                                        Vegetative medium 1                                                           ______________________________________                                        Soluble starch         2% by wt.                                              Gluten meal            1% by wt.                                              Dried yeast            1% by wt.                                              Corn Steep Liquor      1% by wt.                                              Tap water              q.s.                                                   ______________________________________                                    

100 ml. of the medium 1 in each of ten 500 ml. flasks were sterilised inconventional manner and then inoculated with a loopful of culture from astock slant of Streptomyces olivaceogriceus ATCC 31427. The organism wasgrown in the medium at 30° C. for 48 hours on a shaker.

Into a 30-liter Jar-fermentor, there were placed 20 liters of thevegetative medium 2 prepared from the following ingredients:

    ______________________________________                                        Vegetative medium 2                                                           ______________________________________                                        Soluble starch         2% by wt.                                              Cottonseed meal        0.5% by wt.                                            Gluten meal            0.5% by wt.                                            Dried yeast            0.5% by wt.                                            Corn Steep Liquor      0.5% by wt.                                            Tap water              q.s.                                                   ______________________________________                                    

The vegetative medium 2 (pH 7.0) was sterilised in a conventional mannerand then inoculated aseptically with the whole volume of the vegetativeinoculum culture prepared above. The organism was grown in the medium 2°at 30° C. for 24 hours.

The whole volume of the vegetative inoculum thus prepared wasaseptically inoculated into a 2000-liter fermentor, containing 1600liters of the fermentation medium prepared from the followingingredients:

    ______________________________________                                        Fermentation medium                                                           ______________________________________                                        Soluble starch       2% by wt.                                                Cottonseed meal      0.5% by wt.                                              Wheat germ           0.5% by wt.                                              Dried yeast          0.25% by wt.                                             Corn Steep Liquor    0.25% by wt.                                             KH.sub.2 PO.sub.4    0.5% by wt.                                              Na.sub.2 HPO.sub.4.12 H.sub.2 O                                                                    0.5% by wt.                                              CoCl.sub.2.6 H.sub.2 O                                                                             1.75 mg/l                                                Tap water            q.s.                                                     ______________________________________                                    

The organism was cultured in the fermentation medium for 72 hours at 30°C. During the growth period, the broth was stirred with a propelleroperating at 170 r.p.m. and sterile air was passed through the broth ata rate of 1600 liters per minute.

After the fermentation was completed, 20 kg "Radiolite" (trade name, afilter aid material sold by Showa Chemical Company, Japan) was added tothe culture broth and the mixture was filtered to remove mycelia. 1600liters of the filtrate was passed through a column of activated charcoal(800 liters) and then was washed with 1600 liters of water. Elution wascarried out with 3000 liters of 50% aqueous acetone and then the eluatewas concentrated to a volume of about 600 liters.

The concentrate was passed through a column of DEAE-Sephadex (tradename, made by Pharmacia A.B.) (200 liters) which has previously beenbuffered with phosphate buffer (pH 6.0). The column was successivelywashed with 200 liters of water and 200 liters of 0.1M sodium chloridesolution and then eluted with 400 liters of 0.3M sodium chloridesolution. The aqueous eluate was passed through a column of an activatedcharcoal (200 l), washed with 200 liters of water and then eluted with400 liters of 50% aqueous acetone. The eluate was concentrated and thenfreeze-dried to give 800 g of a white powder. The powder was dissolvedinto 25 liters of water and the solution was passed through a column ofCM-Sephadex (H⁺ form)(25 l). The column was eluted with 25 liters ofwater and the eluate was concentrated and then freeze-dried to give 33g. of yellowish white powder. The powder was placed on the top of acolumn of cellulose (1.2 l). The column was washed with 1000 ml. of 70%aqueous propanol and then eluted with 1000 ml. of 60% aqueous propanol.The eluate was concentrated and freeze-dried to give 4 g. of whitepowder. The powder was dissolved into 150 ml. of water and then thesolution was subjected to column chromatography on Sephadex G-15 (2.8l).

The column was developed and eluted with water. The active fractionswere collected and concentrated and then freeze-dried to give 4 g. of awhite powder. The powder dissolved into 25 ml. of water and the solutionwas subjected to column chromatography on CM-Sephadex (H⁺ form)(400 ml).The column was developed and eluted with water. The active fractionswere collected, concentrated and then freeze-dried to give 40 mg. of theFR-900156 substance in the form white powders (purity: about 70%).

(2) Fermentation was carried out in the same manner as described inExample 94 (1). After the fermentation was completed, 20 kg. "Radiolite"(trade.name, a filter aid material sold by Showa Chemical Company) wasadded to the culture broth and the mixture was filtered to removemycelia. 1600 Liters of the filtrate was passed through a column ofactivated charcoal (800 liters) and then was washed with 1600 liters ofwater. Elution was carried out with 3000 liters of 50% aqueous acetoneand then the eluate was concentrated to a volume of about 600 liters.The concentrate was passed through a column of DEAE-Sephadex (tradename, made by Pharmacia A.B.)(200 liters) which has previously beenbuffered with phosphate buffer (pH 6.0). The column was successivelywashed with 200 liters of water and 200 liters of 0.1M sodium chloridesolution and then eluted with 400 liters of 0.3M sodium chloridesolution. The aqueous eluate was passed through a column of an activatedcharcoal (200 liters), washed with 200 liters of water and then elutedwith 400 liters of 50% aqueous acetone. The eluate was concentrated andthen freeze-dried to give 800 g. of white powder. The powder wasdissolved into 25 liters of water and the solution was passed through acolumn of CM-Sephadex (H⁺ form)(25 liters). The column was eluted with25 liters of water and the eluate was concentrated and then freeze-driedto give 33 g. of yellowish white powders. The powders were placed on topof a column of cellulose (1.2 liters). The column was washed with 1000ml. of 70% aqueous propanol and then eluted with 1000 ml. of 60% aqueouspropanol. The eluate was concentrated and freeze-dried to give 4 g. ofwhite powder. The powder was dissolved into 300 ml. of water and thenpassed through a column of DEAE-Sephadex (trade name, made by PharmaciaA.B.) (1.4 liters), which has previously been buffered with phosphatebuffer (pH 6.0). The column was washed with 1.5 liters of 0.1M sodiumchloride solution and then eluted with 3 liters of 0.2M sodium chloridesolution. The active fractions were collected and then passed through acolumn of activated charcoal (300 ml). The column was washed with waterand then eluated with 800 ml. of 50% aqueous acetone. The eluate wasconcentrated and then freeze-dried to give 700 mg. of white powders. Thepowders were dissolved into 20 ml. of water and then passed through acolum of CM-Sephadex (H⁺ form)(500 ml). The column was eluted with waterand the active fractions were collected, and then concentrated to give10 ml. of concentrate. The concentrate was subjected to a columnchromatography on Sephadex G 15 (500 ml) and developed with water. Theactive fractions were collected and concentrated and then freeze-driedto give 70 mg. of white powders. The powders were dissolved into 25 ml.of water and subjected to preparative thin layer chromatography oncellulose (made by Eastman Kodak Co.): a developing solvent was mixtureof butanol, acetic acid and water (4:1:2). Elution was carried out with50 ml. of water and the eluate was concentrated and then freeze-dried togive 20 mg of the FR-900156 substance in the form of a white powder.

(3) Fermentation was carried out in the same manner as described inExample 94 (1). After the fermentation was completed, 20 kg. "Radiolite"(trade name, a filter aid material sold by Showa Chemical Company) wasadded to the culture broth and the mixture was filtered to removemycelia. 1600 Liters of the filtrate was passed through a column ofactivated charcoal (800 liters) and then was washed with 1600 liters ofwater. Elution was carried out with 3000 liters of 50% aqueous acetoneand then the eluate was concentrated to a volume of about 600 liters.The concentrate was passed through a column of DEAE-Sephadex (tradename, made by Pharmacia A.B.)(200 liters) which has previously beenbuffered with phosphate buffer (pH 6.0). The column was successivelywashed with 200 liters of water and 200 liters of 0.1M sodium chloridesolution and then eluted with 400 liters of 0.3M sodium chloridesolution. The aqueous eluate was passed through a column of an activatedcharcoal (200 liters), washed with 200 liters of water and then elutedwith 400 liters of 50% aqueous acetone. The eluate was concentrated andthen freeze-dried to give 800 g. of white powder. The powder wasdissolved into 25 liters of water and the solution was passed through acolumn of CM-Sephadex (H⁺ form) (25 liters). The column was eluted with25 liters of water and the eluate was concentrated and then freeze-driedto give 33 g. of yellowish white powder. The powder was placed on top ofa column of cellulose (1.2 liters). The column was washed with 1000 ml.of 70% aqueous propanol and then eluted with 1000 ml. of 60% aqueouspropanol. The eluate was concentrated and freeze-dried to give 4 g. ofwhite powder. The powder was dissolved into 300 ml. of water and thenpassed through a column of DEAE-Sephadex (trade name, made by PharmaciaA.B.)(1.4 liters) which has previously been buffered with phosphatebuffer (pH 6.0). The column was washed with 1.5 liters of 0.1M sodiumchloride solution and then eluted with 300 liters of 0.2M sodiumchloride solution. The active fractions were collected and then passedthrough a column of activated charcoal (300 ml). The column was washedwith water and then eluated with 800 ml. of 50% aqueous acetone. Theeluate was concentrated and then freeze-dried to give 700 mg. of whitepowders. The powders were dissolved into 20 ml. of water and then passedthrough a column of CM-Sephadex (H⁺ form)(500 ml). The column was elutedwith water and the active fractions were collected, and thenconcentrated to give 10 ml. of concentrate. The concentrate wasfreeze-dried to give 400 mg of a powder. The powder was placed on thetop of a column of cellulose (500 ml). Elution was carried out with amixture of n-butanol, acetic acid and water (4:1:2). The activefractions were collected and freeze-dried to give 200 mg of a powder.The powder was dissolved into 7 ml. of water and then placed on SephadexG 15 (250 ml).

The active fractions were collected, concentrated and then freeze-driedto give 100 mg of the FR-900156 substance in the form of a white powder.

(4) A white powder of FR-900156 substance obtained by the Example 94 (3)was further purified by conducting repeatedly the above purificationmeans to give a more purified product of FR-900156 substance.

(5) 100 ml of a medium containing corn starch 2% (by wt.), gluten meal1%, dried yeast 1% and corn steep liquor 1% were poured into each of ten500 ml flasks and sterilised in a conventional manner and theninoculated with a loopful of culture from a stock slant of Streptomycesviolaceus ATCC 31481.

The organism was grown in the medium at 30° C. for 48 hours on a shaker.

Into a 30-liters Jar-fermentor, there were placed 20-liters of the samemedium as above. The medium was sterilised in a conventional manner andthen inoculated aseptically with the whole volume of the inoculumculture prepared above. The organism was grown in the medium at 30° C.for 30 hours.

The whole volume of the inoculum thus prepared was asepticallyinoculated into a 400-liters fermentor, containing 320 liters of themedium (pH 6.5) containing soluble starch 2% (by wt.), gluten meal 1%,cottonseed meal 1% and sodium sulfate (10 hydrates) 2%.

The organism was cultured in the medium at 30° C. for 72 hours. Duringthe growth period, the broth was stirred with a propeller operating at170 r.p.m. and sterile air was passed through the broth at a rate of 320liters per minute. After the fermentation was complete, 4 kg of"Radiolite" was added to the cultured broth and the mixture was filteredto remove mycelia. 300 liters of the filtrate was passed through acolumn of activated charcoal (150 liters) and then washed with 300liters of water. Elution was carried out with 600 liters of 50% aqueousacetone and then the eluate was concentrated to a volume of about 120liters.

The concentrate was passed through a column of DEAE-Sephadex (30 liters)which has previously been buffered with phosphate buffer (pH 6.0). Thecolumn was successively washed with 30 liters of water and 30 liters of0.1M sodium chloride and then eluted with 0.3M sodium chloride. Theeluate (60 liters) was passed through a column of an activated charcoal(30 liters), washed with 60 liters of water and then eluted with 60liters of 50% aqueous acetone. The eluate was freeze-dried to give 120 gof a white powder. The powder was dissolved in 4 liters of water and thesolution was passed through a column of CM-Sephadex (H⁺ form)(14liters). The column was eluted with water and the eluate wasconcentrated and then freeze-dried to give 20 g of a powder. The powderwas placed on the top of the column of cellulose. Elution was carriedout with an aqueous propanol and the active fractions were collected andfreeze-dried to give 5 g of a powder. The powder was dissolved in 500 mlof water and the solution was passed through a column of DEAE-Sephadex(1.3 liters) which has previously been buffered with phosphate buffer(pH 6). The column was washed with 0.1M sodium chloride and eluted with0.2M sodium chloride. The active fractions were collected and passedthrough a column of an activated charcoal (900 ml). The column waswashed with water and eluted with 50% aqueous acetone (200 ml). Theeluate was concentrated and freeze-dried to give 1 g of a powder.

The powder was dissolved in 300 ml of water and the solution was passedthrough a column of CM-Sephadex (H⁺ form)(250 ml). The column wasdeveloped and eluted with water. The active fractions were collected,concentrated and then freeze-dried to give 800 mg of a powder. Thepowder was dissolved in 20 ml of water and the solution was mixed with asmall amount of cellulose. The mixture was subjected to columnchromatograph on cellulose. The column was washed successively with 100ml of acetone and 300 ml of mixture of n-butanol: acetic acid: water(4:1:1) and then developed and eluted with a mixture of n-butanol:acetic acid: water (4:1:2)(1 liter). The active fractions were collectedand freeze-dried to give 30 mg of a powder. The powder was dissolved in20 ml of water and the solution was passed through a column of SephadexG 15 (250 ml). The column was developed and eluted with water and theactive fractions were collected and then freeze-dried to give 5 mg ofFR-900156 substance.

EXAMPLE 95: for the pharmaceutical composition

(1) Preparation for injection

The required quantities of the FR-900156 substance were distributed intovials, each containing 500 mg of the active ingredient. The vials weresealed hermetically to exclude bacteria. Whenever the vial is requiredfor use, 2 ml of sterile distilled water for injection is added to thevial and then the aqueous solution is administered by injection.

(2) Preparation of tablet

A suitable formulation for a tablet consists of the following mixture.

    ______________________________________                                        FR-900156 substance    200    mg                                              Mannitol               400    mg                                              Starch                 50     mg                                              Magnesium stearate     10     mg                                              ______________________________________                                    

(3) Preparation of capsule

    ______________________________________                                        FR-900156 substance    300    mg                                              Magnesium stearate     15     mg                                              ______________________________________                                    

The above ingredients were mixed and then inserted into a hard gelatincapsule in a conventional manner.

Preparation 66

(1) Step 1 ##STR392##

Z-(L)-mesoDAP-(L)-D-AlaOH (1)(1.80 g) was suspended in a mixture ofmethylene chloride (50 ml), bis(trimethylsilyl)acetamide (5.60 g) anddimethylformamide (6 ml). The suspension was stirred for 4 hours atambient temperature and treated with trifluoroacetic anhydride (1.0 g)and further stirred for an hour. The reaction mixture was concentratedunder reduced pressure and the residual oil was extracted with ethylacetate. The organic layer was washed with water, dried over magnesiumsulfate and then evaporated to give Z-(L)-Tfa-(D)-mesoDAP-(L)-D-AlaOH(2).

NMR (DMSO-d₆), δ(ppm): 1.18 (3H, d, J=7 Hz), 1.10-2.0 (6H, m), 3.90-4.30(3H, m), 5.03 (2H, s), 7.33 (5H, s), 8.18 (1H, d, J=7 Hz), 9.55 (1H, d,J=7 Hz).

(2) Step 2 ##STR393##

Tfa-(D)-mesoDAP-(L)-D-AlaOH (3) was prepared in substantially the samemanner as Preparation 25 from compound (2).

IR (Nujol): 3250, 2600-2400 (broad), 1700, 1670 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.33 (3H, d, J=7 Hz), 1.20-2.20 (6H, m), 3.83-4.43(3H, m).

Preparation 67

(1) Step 1 ##STR394##

Z-(L)-Tfa-(D)-mesoDAP-(L)-GlyOH (2) was prepared in substantially thesame manner as step 1 of Preparation 66 from compound (1).

NMR (DMSO-d₆), δ(ppm): 1.10-2.10 (6H, m), 3.73 (2H, d, J=7 Hz),3.90-4.50 (2H, m), 5.06 (2H, s), 7.33 (5H, s), 7.0-7.30 (1H, broad),8.13 (1H, t, J=7 Hz), 9.56 (1H, d, J=7 Hz). (2) Step 2 ##STR395##

Tfa-(D)-mesoDAP-(L)-GlyOH (3) was prepared in substantially the samemanner as Preparation 25 from compound (2).

IR (Nujol): 3250 (shoulder), 2600-2300 (broad), 1720 (shoulder), 1700,1680 (shoulder), 1210, 1180, 1150 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.20-2.20 (6H, m), 3.90 (2H, s), 4.00-4.50 (2H, m).

Preparation 68 ##STR396##

D-Glu(α-oBzl)(1)(4.74 g) was added to a mixture of methylene chloride(50 ml) and bis(trimethylsilyl)acetamide (4.10 g). The mixture wasstirred until a clear solution was obtained and n-octanoyl chloride(3.17 g) was added dropwise thereto. The mixture was reacted for an hourat ambient temperature and then evaporated to give an oily residue towhich water was added. The mixture was extracted with ethyl acetate andthe extract was washed with water, dried over magnesium sulfate and thenevaporated to give a crystalline residue (7.10 g). The residue wasrecrystallized from diisopropyl ether to give n-octanoyl-D-Glu(OH)oBzl(2)(6.0 g).

NMR (CDCl₃), δ(ppm): 0.87 (3H, t, J=7 Hz), 1.0-2.60 (16H, m), 4.50-5.10(1H, m), 5.20 (2H, s), 6.35 (1H, d, J=7 Hz), 7.40 (5H, s), 9.90 (1H,broad s).

The following compounds were prepared in substantially the same manneras Preparation 68.

Preparation 69

n-Heptanoyl-D-Glu(OH)oBzl

NMR (CDCl₃), δ(ppm): 0.86 (3H, t, J=7 Hz), 1.0-2.60 (14H, m), 4.50-5.10(1H, m), 5.16 (2H, s), 6.45 (1H, d, J=7 Hz), 7.35 (5H, s), 10.00 (1H,s).

Preparation 70

Phenoxyacetyl-D-Glu(OH)oBzl

NMR (CDCl₃), δ(ppm): 1.80-2.56 (4H, m). 4.50 (2H, s), 4.50-5.10 (1H, m),5.20 (2H, s), 6.80-7.20 (5H, m), 7.35 (5H, s), 9.40 (1H, broad s).

Preparation 71

D-Acetylmandelyl-D-Glu(OH)oBzl

NMR (CDCl₃), δ(ppm): 1.90-2.50 (4H, m), 2.10 (3H, s), 4.20-4.80 (1H, m),5.10 (2H, s), 6.00 (1H, s), 7.20-7.50 (10H, m).

Preparation 72

n-Hexanoyl-D-Glu(OH)oBzl

NMR (CDCl₃), δ(ppm): 0.90 (3H, t, J=7 Hz), 1.0-2.70 (2H, m), 4.50-5.0(1H, m), 5.17 (2H, s), 6.50 (1H, d, J=7 Hz), 7.37 (5H, s), 10.60 (1H,s).

Preparation 73

N-Phenylcarbamoyl-D-Glu(OH)oBzl

NMR (DMSO-d₆), δ(ppm): 1.70-2.50 (4H, m), 4.15-4.60 (1H, m) 5.13 (2H,s), 6.57 (1H, d, J=8 Hz), 6.70-7.60 (10H, m), 8.50 (1H, s).

Preparation 74

O-Benzylsalicyloyl-D-Glu(OH)oBzl

NMR (CDCl₃), δ(ppm): 0.83-2.30 (26H, m), 2.83 (2H, broad s), 4.70-5.0(1H, m), 5.13 (2H, s), 5.20 (2H, s), 7.0-7.68 (14H, m).

Preparation 75

D-Lac(oAc)-D-Glu(OH)oBzl dicyclohexylamine salt

NMR (CDCl₃), δ(ppm): 0.80-2.40 (26H, m). 1.43 (3H, d, J=7 Hz), 2.10 (3H,s), 2.90 (2H, broad s), 4.40 (1H, q, J=7 Hz), 5.17 (2H, s), 5.17 (1H, q,J=7 Hz), 7.40 (5H, s), 7.90 (1H, d, J=7 Hz), 9.10 (1H, s).

Preparation 76

Nicotinoyl-D-Glu(OH)oBzl

NMR (DMSO-d₆), δ(ppm): 1.83-2.67 (4H, m). 4.40-4.85 (1H, m), 5.20 (2H,s), 7.40 (5H, s), 7.50-7.70 (1H, m), 8.10-8.40 (1H, m), 8.67-9.20 (3H,m).

Preparation 77 ##STR397##

To a mixture of bis(trimethylsilyl)acetamide (2.4 ml) and methylenechloride (25 ml) was added Boc-(L)-mesoDAP-(D)-NHNHZ (1)(1.0 g). Theresulting mixture was stirred for two hours at ambient temperature andcooled to 15° C. and then carbobenzyl chloride (505 mg) was addedthereto.

The reaction mixture was stirred for an hour at ambient temperature andconcentrated to about 5 ml. To the concentrate were added ethyl acetate(30 ml) and 2% hydrochloric acid (10 ml). Ethyl acetate layer wasseparated and washed with water and dried over magnesium sulfate. Ethylacetate was evaporated to dryness under reduced pressure and the residuewas pulverized with isopropylether to giveBoc-(L)-Z-(D)-mesoDAP-(D)-NHNHZ (2)(0.60 g).

NMR (CD₃ OD), δ(ppm): 1.43 (9H, s), 1.3-2.1 (6H, m), 3.9-4.3 (2H, m),5.08 (2H, s), 5.13 (2H, s), 7.32 (10H, s).

Preparation 78 ##STR398##

Boc-(L)-Z-(D)-mesoDAP-(D)-NHNHZ (1)(500 mg) was dissolved intrifluoroacetic acid (5 ml) and the solution was stirred for 15 minutesat ambient temperature. After evaporation of trifluoroacetic acid, theresidue was dissolved in water (3 ml) and neutralized with 5% sodiumbicarbonate. The resulting crystal was filtered and washed with water togive Z-(D)-mesoDAP-(D)-NHNHZ (2)(280 mg), which was identical with thatof product prepared in Preparation 62.

Preparation 79 ##STR399##

To a solution of 50% aqueous dioxane (20 ml) were addedZ-(D)-mesoDAP-(D)-NHNHZ (1)(500 mg) and triethylamine (250 mg).

Di-tert-butyldicarbonate (300 mg) was added to the mixture. Theresulting mixture was stirred for three hours at ambient temperature andthen evaporated. The resulting aqueous solution was acidified to pH 3with 5% hydrochloric acid and extracted with ethyl acetate (30 ml).Ethyl acetate layer was separated and the residue was washed with water,dried over magnesium sulfate. Ethyl acetate was evaporated to drynessunder reduced pressure and the residue was pulverized with isopropylether to give Boc-(L)-Z(D)-mesoDAP-(D)-NHNHZ (2)(0.47g), which wasidentified with that of the product prepared in Preparation 77.

Preparation 80

(1) Step 1 ##STR400##

Z-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-γ-AbuoBzl (2) was prepared insubstantially the same manner as step 1 of Preparation 43 from compound(1).

NMR (DMSO-d₆), δ(ppm): 0.9-2.0 (26H, m), 2.3 (2H, m), 2.9-3.4 (2H, m),3.6-4.2 (2H, m), 5.02 (2H, s), 5.09 (2H, s), 6.7 (1H, m), 7.2 (1H, m),7.35 (10H, s), 7.80 (1H, m), 8.65 (1H, m), 9.58 (1H, broad s).

(2) Step 2 ##STR401##

Boc-(D)-mesoDAP-(D)-NHNH.Boc-(L)-γ-AbuOH (3) was prepared insubstantially the same manner as step 2 of Preparation 43 from compound(2).

NMR (D₂ O), δ(ppm): 1.0-2.6 (10H, m), 3.28 (2H, t, J=6.5 Hz), 3.7-4.3(2H, m).

Preparation 81

(1) Step 1 ##STR402##

Z-(L)-Boc-(D)-mesoDAP-(D)-NHNH.Boc-(L)-D-LeuOH (2) was prepared insubstantially the same manner as step 1 of Preparation 43 from compound(1).

NMR (DMSO-d₆), δ(ppm): 0.6-2.0 (33H, m), 3.6-4.6 (3H, m), 5.01 (2H, s),5.10 (2H, s), 6.7 (1H, m), 7.2 (1H, m), 7.34 (10H, s), 8.1-8.9 (2H, m),9.57 (1H, broad s).

(2) Step 2 ##STR403##

Boc-(D)-mesoDAP-(D)-NHNH.Boc-(L)-D-LeuOH (3) was prepared insubstantially the same manner as step 2 of Preparation 43 from compound(2).

IR (Nujol): 1680 (broad) and 1540 (broad) cm⁻¹.

NMR (CD₃ OD), δ(ppm): 1.0 (6H, m), 1.50 (18H, s), 1.2-2.3 (9H, m),3.6-4.6 (3H, m).

Preparation 82 ##STR404##

Heptanoyl-β-D-Asp(OH)oBzl (3) was prepared in substantially the samemanner as Preparation 56 from compounds (1) and (2).

NMR (CDCL₃), δ(ppm): 0.7-1.0 (3H, m), 1.0-2.0 (8H, m), 2.95 (t 2H, 4Hz), 4.90 (t,t, 1H, 4 Hz, 8 Hz), 5.14 (2H, s), 6.72 (1H, 8 Hz), 7.25(5H, s), 10.00 (1H, s).

Preparation 83 ##STR405##

Heptanoyl-δ-D-Aad(OH)oBzl (3) was prepared in substantially the samemanner as Preparation 56 from compounds (1) and (2).

(as dicyclohexylamine salt):

NMR (CDCL₃), δ(ppm): 0.7-1.1 (3H, m), 1.1-2.5 (34H), 2.5-3.1 (2H, m),4.3-4.8 (1H, m), 5.13 (2H, s), 6.60 (1H, d, J=8 Hz), 7.33 (5H, s), 8.50(3H, s).

Preparation 84 ##STR406##

Mandelyl(oAc)-δ-D-Aad(OH)oBzl (3) was prepared in substantially the samemanner as Preparation 56 from compounds (1) and (2).

NMR (CDCL₃), δ(ppm): 1.5-2.0 (4H, m), 2.13 (3H, s), 2.0-2.5 (2H, m),4.4-4.8 (1H, m), 5.13 (2H, s), 6.07 (1H, s), 6.7-7.0 (1H, m), 7.33 (10H,s), 9.33 (1H, broad s).

EXAMPLE 96

(1) Step 1 ##STR407##

Phenoxyacetyl-D-Glu(α-oBzl)(1)(742 mg) was dissolved in methylenechloride and N-methylmorpholine (202 mg) was added thereto. Thissolution was cooled under a dryice-carbontetrachloride andisobutylchloroformate (274 mg) was added thereto. The mixture wasallowed to react for 60 minutes at the same temperature. To theresulting reaction mixture was added the solutionBoc-(D)mesoDAP-(D)-NHNHBoc (2)(808 mg) in a mixture of methylenechloride (20 ml), dimethylformamide (2 ml) and bis(trimethylsilyl)acetamide (2 ml). The reaction mixture was stirred fortwo hours and concentrated in vacuo to give an oily residue, which wasdissolved in ethyl acetate, washed with 1N hydrochloric acid and brine,dried over magnesium sulfate. Evaporation of the solvent gave a whitefoam, which was pulverized with isopropylether to give aphenoxyacetyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3)(1.21g).

NMR (CD₃ OD), δ(ppm): 1.42 (18H, s), 3.9-4.6 (3H, m), 4.54 (2H, s), 5.17(2H, s), 6.85-7.30 (5H, m), 7.35 (5H, s).

(2) Step 2 ##STR408##

Phenoxyacetyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3)(1.13g) was dissolved in methanol (20 ml) and 1N sodium hydroxide (3.0 ml)was added thereto at ambient temperature. The reaction mixture wasstirred for 4 hours at the same temperature and concentrated underreduced pressure to give an oily residue, which was dissolved in amixture of ethyl acetate (60 ml), water (10 ml) and 1N hydrochloric acid(4 ml). The organic layer was with brine, dried over magnesium sulfateand then concentrated in vacuo to givephenoxyacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4)(670 mg).

NMR (CD₃ OD), δ(ppm): 1.43 (18H, s), 4.00-4.70 (3H, m), 4.60 (2H, s),7.24-7.50 (5H, m).

(3) Step 3 ##STR409##

Phenoxyacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4)(615 mg)was dissolved in trifluoroacetic acid (3 ml) and the solution was keptfor an hour at ambient temperature. The solvent was distilled off togive an oily paste, which was pulverized with ether to give a whitepowder. The powder was dissolved in a mixture of water (25 ml) and 1Nsulfuric acid and the solution was cooled in an ice-bath. To thesolution was added sodium periodate (300 mg) and the mixture was stirredfor two hours at 0°0 C. The mixture was treated with an aqueous sodiumbisulfite and then passed through a column packed with a macroporousnon-ionic adsorption resin, HP-20. Elution was carried out with amixture of water and methanol (7:3) and the eluate was concentrated invacuo to give a white powder, which was dissolved in water andlyophilized to give phenoxyacetyl-γ-D-Glu(α-OH)-(L)-mesoDAP (5)(150 mg).

NMR (D₂ O+NaHCO₃), δ(ppm): 1.25-2.35 (10H, m), 3.72 (1H, t, J=6 Hz),4.00-4.35 (2H, m), 4.63 (2H, s), 7.0-7.5 (5H, m).

EXAMPLE 97

(1) Step 1 ##STR410##

n-Octanoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(3) was prepared in substantially the same manner as step (1) of Example96 from compounds (1) and (2).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.83 (3H, t, J=6 Hz), 1.37 (18H, s),3.80-4.50 (4H, m), 5.08 (2H, s), 7.33 (5H, s), 7.66-8.33 (6H, m), 9.20(1H, broad s).

(2) Step 2 ##STR411##

n-Octanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (4)was prepared in substantially the same manner as step 2 of Example 96from compound (3).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.87 (3H, t, J=6 Hz), 1.35 (18H, s),4.00-4.66 (4H, m), 7.66-8.33 (6H, m), 9.55 (2H, broad s).

(3) Step 3 ##STR412##

n-Octanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was prepared insubstantially the same manner as step 3 of Example 96 from compound (4).

NMR (D₂ O), δ(ppm): 0.88 (3H, t, J=7 Hz), 1.40 (3H, d, J=7 Hz),1.00-2.50 (22H, m), 3.80 (1H, t, J=7 Hz), 4.00-4.50 (3H, m).

EXAMPLE 98

(1) Step 1 ##STR413##

Phenylcarbamoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared in substantially the same manner as step 1 of Example 96 fromCompounds (1) and (2).

NMR (CD₃ OD) δ(ppm): 1.41 (18H, s), 3.90-4.62 (3H, m), 5.19 (2H, s),6.95-7.52 (10H, m).

(2) Step 2 ##STR414##

Phenylcarbamoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4) wasprepared in substantially the same manner as step (2) of Example 96 fromcompound (3).

NMR (CD₃ OD) δ(ppm): 1.48 (18H, s), 3.95-4.55 (3H, m), 7.32 (5H, s).

(3) Step 3 ##STR415##

Phenylcarbamoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (5) was prepared insubstantially the same manner as step 3 of Example 96 from Compound (4).

NMR (D₂ O+NaOD) δ(ppm): 1.1-2.6 (10H, m), 3.16 (1H, m), 4.00-4.20 (2H,m), 7.00-7.45 (5H, m).

EXAMPLE 99

(1) Step 1 ##STR416##

α-Ethylhexanoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared in substantially the same manner as step 1 of Example 96 fromcompound (1) and (2).

NMR (CD₃ 0D) δ(ppm): 0.86 (6H, m), 1.45 (18H, s), 3.90-4.30 (3H, m),5.16 (2H, s), 7.36 (5H, s).

(2) Step 2 ##STR417##

α-Ethylhexanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4) wasprepared in substantially the same manner as step 2 of Example 96 fromcompound (3).

NMR (CD₃ OD) δ(ppm): (0.88 (6H, m), 1.43 (18H, s), 4.02 (1H, m), 4.36(2H, m).

(3) Step 3 ##STR418##

α-Ethylhexanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (5) was prepared insubstantially the same manner as step 3 of Example 96 from Compound (4).

NMR (D₂ O) δ(ppm): 0.83 (6H, m), 1.08-2.56 (19H, m), 3.82 (1H, t, J=7Hz), 4.23-4.48 (2H, m).

EXAMPLE 100

(1) Step 1 ##STR419##

To a solution of γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (1)(800mg) in the mixture of water (8 ml) and dioxane (8 ml) were addedN-hydroxysuccinylnicotinate (330 mg) and triethylamine (364 mg). Thereaction mixture was left for 20 hours at ambient temperature andconcentrated in vacuo. To the residue was added 1N hydrochloric acid (3ml) and the mixture was passed through a column packed with amacroporous non-ionic adsorption resin, HP 20 (120 ml).

The fraction eluted with a mixture of water and methanol (2:3) wasconcentrated in vacuo to givenicotinoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (2).

NMR (CD₃ OD), δ(ppm): 1.44 (18H, s), 3.9-4.6 (3H, m), 7.59 (1H, d.d.,J=8 and 5 Hz), 8.35 (1H, m), 8.75 (1H, d, J=5 Hz), 9.11 (1H, m).

(2) Step 2 ##STR420##

Nicotinoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (3) was prepared in substantiallythe same manner as Step 2 of Example 96 from Compound (2).

NMR (D₂ O), δ(ppm): 1.25-2.60 (10H, m), 3.87 (1H, t, J=6 Hz), 4.23 (1H,m), 4.51 (1H, m), 7.98 (1H, d.d., J=6 and 8 Hz), 8.71 (1H, d, J=8 Hz),8.88 (1H, d, J=6 Hz), 9.10 (1H, s).

EXAMPLE 101

(1) Step 1 ##STR421##

D-Lac(oAc)-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared in substantially the same manner as Step 1 of Example 59 fromCompounds (1) and (2).

IR (Nujol): 3280, 1730, 1660, 1530 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 1.38 (18H, s), 2.05 (3H, s), 3.85-4.60 (3H, m).

(2) Step 2 ##STR422##

To a solution ofD-Lac(oAc)-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4)(1.14 g)in methanol (15 ml) was added 1N sodium hydroxide (6.2 ml).

The reaction mixture was left for 3.5 hours at ambient temperature and1N hydrochloric acid (6.2 ml) was added thereto. The mixture wasconcentrated in vacuo and the residue was passed a column packed with amacroporous non-ionic adsorption resin, HP 20 (100 ml) and eluted with amixture of water and methanol (1:1). The fractions containing the objectCompound (4) were collected and concentrated in vacuo to giveD-Lac(OH)-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4) (510 mg).

IR (Nujol): 3300, 1720(sh), 1650, 1520 cm⁻¹.

NMR (CD₃ OD), δ(ppm): 1.45 (18H, s), 3.97-4.70 (4H, m).

(3) Step 3 ##STR423##

D-Lac(OH)-γ-D-Glu(α-OH)-(L)-mesoDAP (5) was prepared in substantiallythe same manner as Step 3 of Example 59 from Compound (4).

IR (Nujol): 3250 (sh), 1720, 1650, 1530 cm⁻¹.

NMR (D₂ O), δ(ppm): 1.25-2.50 (10H, m), 1.36 (3H, d, J=7 Hz), 3.91 (1H,t, J=7 Hz), 4.28-4.45 (3H, m).

EXAMPLE 102

(1) Step 1 ##STR424##

α-Ethylhexanoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(3) was prepared in substantially the same manner as step 1 of Example96 from compounds (1) and (2).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.80-1.00 (6H, m), 1.40 (18H, s), 1.00-2.33(22H, m), 4.00-4.50 (4H, m), 5.10 (2H, s), 7.33 (5H, s), 7.66-8.33 (6H,m), 9.50 (1H, broad s).

(2) Step 2 ##STR425##

α-Ethylhexanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(4) was prepared in substantially the same manner as step 2 of Example96 from compound (3).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.83-1.00 (6H, m), 1.43 (18H, s), 1.00-2.33(22H, m), 3.50-4.66 (4H, m), 7.66-8.33 (6H, m), 9.53 (2H, broad s).

(3) Step 3 ##STR426##

α-Ethylhexanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was preparedin substantially the same manner as step 3 of Example 96 from compound(4).

NMR (D₂ O), δ(ppm): 0.84 (6H, t, J=7 Hz), 1.24-2.30 (19H, m), 1.36 (3H,t, J=7 Hz), 3.82 (1H, t, J=6 Hz), 4.25-4.44 (3H, m).

EXAMPLE 103

(1) Step 1 ##STR427##

Hexanoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (3)was prepared in substantially the same manner as step 1 of Example 96from compound (1) and (2).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.83 (3H, t, J=7 Hz), 1.44 (18H, s),3.83-4.50 (4H, m), 5.13 (2H, s), 7.33 (5H, s), 7.73-8.73 (5H, m), 9.56(1H, broad s).

(2) Step 2 ##STR428##

Hexanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (4)was prepared in substantially the same manner as step 2 of Example 96.

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.86 (3H, t, J=7 Hz), 1.37 (18H, s),3.83-4.33 (4H, m), 7.66-8.77 (6H, m).

(3) Step 3 ##STR429##

Hexanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was prepared insubstantially the same manner as step 3 of Example 96 from compound (4).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 0.80 (3H, t, J=7 Hz), 1.38 (5H, d, J=7 Hz),1.28-2.40 (18H, m), 3.75 (1H, t, J=7 Hz), 4.20-4.35 (3H, m).

EXAMPLE 104

(1) Step 1 ##STR430##

Phenoxyacetyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(3) was prepared in substantially the same manner as step 1 of Example96 from compound (1) and (2).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 1.38 (18H, s), 3.66-4.50 (4H, m), 4.53 (2H,s), 5.15 (2H, s), 6.87-7.33 (5H, m), 7.37 (5H, s), 7.85-8.60 (6H, m),9.57 (1H, broad s).

(2) Step 2 ##STR431##

Phenoxyacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(4) was prepared in substantially the same manner as step 2 of Example96 from compound (3).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 1.40 (18H, s), 3.73-4.50 (4H, m), 4.53 (2H,s), 6.90-7.43 (5H, m), 7.73-8.66 (6H, m), 9.50 (2H, broad s).

(3) Step 3 ##STR432##

Phenoxyacetyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was prepared insubstantially the same manner as step 3 of Example 96 from compound (4).

NMR (D₂ O), δ(ppm): 1.39 (3H, d, J=7 Hz), 1.60-2.50 (10H, m), 3.85 (1H,t, J=7 Hz), 4.00-4.50 (3H, m), 4.64 (2H, s), 6.80-7.50 (5H, m).

EXAMPLE 105

(1) Step 1 ##STR433##

Phenylcarbamoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(3) was prepared in substantially the same manner as step 1 of Example96 from compounds (1) and (2).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 1.37 (18H, s), 3.83-4.50 (4H, m), 5.13 (2H,s), 7.33 (5H, s), 6.50-7.20 (5H, m), 7.33-8.50 (7H, m), 9.50 (1H, broads).

(2) Step 2 ##STR434##

Phenylcarbamoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(4) was prepared in substantially the same manner as step 2 of Example96 from compound (3).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 1.42 (18H, s), 3.83-4.20 (4H, m), 6.40-7.50(5H, m), 7.66-8.60 (7H, m), 9.50 (2H, broad s).

(3) Step 3 ##STR435##

Phenylcarbamoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was preparedin substantially the same manner as step 3 of Example 96.

NMR (D₂ O), δ(ppm): 1.40 (5H, d, J=7 Hz), 1.30-2.50 (10H, m), 3.76 (1H,t, J=7 Hz), 4.20-4.50 (3H, m), 7.00-7.70 (5H, m).

EXAMPLE 106

(1) Step 1 ##STR436##

D-Lac(oAc)-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH(3) was prepared in substantially the same manner as step 1 of Example96 from compounds (1) and (2).

NMR (DMSO-d₆ +D₂ O), δ(ppm): 1.40 (18H, s), 2.06 (3H, s), 1.10-2.30(16H, m), 3.66-4.57 (5H, m), 5.13 (2H, s), 7.37 (5H, s), 7.66-8.38 (6H,m), 9.56 (1H, broad s).

(2) Step 2 ##STR437##

D-Lac(OH)-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (4)was prepared in substantially the same manner as step 2 of Example 96from compound (3).

NMR (CD₃ OD), δ(ppm): 1.40 (9H, s), 1.43 (9H, s), 1.33-2.50 (16H, m),3.83-4.66 (5H, m).

(3) Step 3 ##STR438##

D-Lac(OH)-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was prepared insubstantially the same manner as step 3 of Example 96 from compound (4).

NMR (D₂ O), δ(ppm): 1.32 (3H, d, J=7 Hz), 1.33 (3H, d, J=7 Hz),1.20-2.40 (10H, m), 3.82 (1H, t, J=7 Hz), 4.20-4.44 (4H, m).

EXAMPLE 107

(1) Step 1 ##STR439##

Z-L-Ala-γ-D-Glu(α-oBzl)-(L)-Tfa-(D)-mesoDAP-(L)-D-AlaOH (3) was preparedin substantially the same manner as step 1 of Example 32 from compounds(1) and (2).

NMR (DMSO-d₆), δ(ppm): 1.23 (6H, d, J=7 Hz), 1.20-2.30 (10H, m),4.0-4.63 (5H, m), 5.07 (2H, s), 5.15 (2H, s), 7.40 (5H, s), 7.73-8.50(3H, m), 9.63 (1H, d, J=7 Hz).

(2) Step 2 ##STR440##

L-Ala-γ-D-Glu(α-OH)-(L)-Tfa-(D)-mesoDAP-(L)-D-AlaOH (4) was prepared insubstantionally the same manner as step 2 of Example 32 from compound(3).

NMR (D₂ O), δ(ppm): 1.43 (3H, d, J=7 Hz), 1.60 (3H, d, J=7 Hz),1.40-2.70 (10H, m), 4.00-4.50 (5H, m).

(3) Step 3 ##STR441##

L-Ala-γ-D-Glu(α-OH)-(L)-Tfa-(D)-mesoDAP-(L)-D-Ala(OH) (4) (810 mg) wasdissolved in a mixture of methylene chloride (30 ml), methanol (3 ml)and triethylamine (594 mg). To this solution was added retinoic acidN-hydroxysuccimide ester (584 mg) and the mixture was reacted for twodays at ambient temperature. Triethylamine (150 mg) was added to thereaction mixture and reacted for another day further. The resultingmixture was evaporated and the oily residue was extracted with ethylacetate after acidification with 1N sulfuric acid. The organic layer waswashed with water, dried over magnesium sulfate and evaporated to give ayellow foamyl residue (1.10 g) which was throughly washed with a mixtureof ethyl ether and isopropylether to giveretinoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Tfa-(D)-mesoDAP-(L)-D-AlaOH (5) (940mg.).

NMR (DMSO-d₆), δ(ppm): 1.00 (6H, 1.03 (3H, d, J=7 Hz), 1.23 (3H, d, J=7Hz), 1.0-2.40 (16H, m), 1.66 (3H, s), 1.93 (3H, s), 2.25 (3H, s),4.0-4.50 (5H, m), 5.83-6.50 (5H, m), 6.70-7.30 (1H, m), 7.70-8.40 (4H,m).

(4) Step 4 ##STR442##

Retinoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Tfa-(D)-mesoDAP-(L)-D-AlaOH (5) (0.90g) was dissolved in a mixture of water (15 ml) and 1N sodium hydroxide(4.50 ml). The resulting solution was maintained for 2 hours at ambienttemperature and then acidified with 1N sulfuric acid. The mixture wasextracted with n-butanol (100 ml). The organic layer was washed threetimes with water (20 ml) and evaporated to give a yellow solid residuewhich was throughly washed with ethyl ethre to giveretinoyl-L-Ala-{-D-Glu(α-OH)-(L)- mesoDAP-(L)-D-AlaOH (6).

NMR (DMSO-d₆), δ(ppm): 1.06 (6H, s), 1.20 (3H, d, J=7 Hz), 1.30 (3H, d,J=7 Hz), 1.73 (3H, s), 2.00 (3H, s), 2.30 (3H, s), 1.00-2.50 (16H, m),3.30-3.70 (1H, m), 4.00-4.66 (4H, m), 5.86-6.50 (5H, m), 6.70-7.30 (1H,m), 7.70-8.30 (4H, m).

EXAMPLE 108

(1) Step 1 ##STR443##

Z-L-Ala-γ-D-Glu(α-oBzl)-(L)-Tfa-(D)-mesoDAP-(L)-GlyOH (3) was preparedin substantially the same manner as step 1 of Example 107 from compounds(1) and (2).

NMR (DMSO-d₆), δ(ppm): 1.23 (3H, d, J=7 Hz), 1.20-2.50 (10H, m), 3.75(2H, d, J=6 Hz), 4.0-4.60 (4H, m), 5.03 (2H, s), 5.13 (2H, s), 7.10-7.30(1H, broad), 7.33 (10H, s), 7.83-8.50 (3H, m), 9.60 (1H, d, J=7 Hz).

(2) Step 2 ##STR444##

L-Ala-γ-D-Glu(α-OH)-(L)-Tfa-(D)-mesoDAP-(L)-GlyOH (4) was prepared insubstantially the same manner as step 2 of Example 107 from compound(3).

NMR (D₂ O), δ(ppm): 1.56 (3H, d, J=7 Hz), 1.20-2.63 (10H, m), 3.95 (2H,s), 4.00-4.50 (4H, m).

(3) Step 3 ##STR445##

Retinoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Tfa-(D)-mesoDAP-(L)-GlyOH (5) wasprepared in substantially the same manner as step 3 of Example 107 fromcompound (4).

NMR (DMSO-d₆), δ(ppm): 1.00 (6H, s), 1.06 (3H, d, J=7 Hz), 1.00-2.40(16H, m), 1.66 (3H, s), 1.90 (3H, s), 2.33 (3H, s), 3.70 (2H, broad),4.0-4.50 (4H, m), 5.83-6.50 (5H, m), 6.70-7.30 (1H, m), 7.70-8.40 (4H,m).

(4) Step 4 ##STR446##

Retinoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (6) was prepared insubstantially the same manner as step 4 of Example 107 from compound(5).

NMR (DMSO-d₆), δ(ppm): 1.00 (6H, s), 1.23 (3H, d, J=7 Hz), 1.70 (3H, s),1.96 (3H, s), 2.26 (3H, s), 1.00-2.50 (16H, m), 3.50 (1H, broad s), 3.76(2H, broad s), 4.0-4.50 (4H, m), 5.70-7.30 (6H, m), 7.70-8.30 (4H, m).

EXAMPLE 109

(1) Step 1 ##STR447##

Nicotinoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-AlaOH (2)was prepared in substantially the same manner as step 1 of Example 100from compound (1).

NMR (D₂ O), δ(ppm): 1.43 (18H, s), 1.35-2.66 (13H, m), 3.87 (1H, t, J=7Hz), 4.00-4.66 (3H, m), 7.90 (1H, m), 8.50-9.20 (3H, m).

(2) Step 2 ##STR448##

Nicotinoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (3) was prepared insubstantially the same manner as step 2 of Example 100 from compound(2).

NMR (NaOD-D₂ O), δ(ppm): 1.33 (3H, d, J=8 Hz), 1.20-2.50 (10H, m), 3.20(1H, t, J=6 Hz), 4.04-4.60 (3H, m), 7.64 (1H, dd, J=4 and 8 Hz), 8.33(1H, dd, J=2 and 8 Hz), 8.72 (1H, dd, J=2 and 4 Hz), 8.94 (1H, dd,J=J=1.5 and 2 Hz).

EXAMPLE 110

(1) Step 1 ##STR449##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Z-(D)-mesoDAP-(D)-NHNHZ-(L)-GlyOH(3) prepared in substantially the same manner as step 1 of Example 96from compounds (1) and (2).

NMR (CD₃ OD), δ(ppm): 1.36 (3H, d, J=7 Hz), 1.44 (3H, d, J=7 Hz), 2.07(3H, s), 1.3-2.4 (10H, m), 3.92 (2H, s), 4.1-4.6 (4H, m), 5.07 (2H, s),5.13 (2H, s), 5.15 (2H, s), 7.32 (15H, s).

(2) Step 2 ##STR450##

D-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Z-(D)-mesoDAP-(D)-NHNHZ-(L)-GlyOH(3)(180 mg) was hydrogenated in acetic acid (25 ml) over 10% palladiumblack (150 mg) for 3.5 hours under two atomospheric pressure of hydrogenat ambient temperature. After completion of the reaction, the catalystwas filtered off and the filtrate was evaporated to dryness underreduced pressure and then the residue was dissolved in water (10 ml). Tothe solution was added 1N hydrochloric acid (0.4 ml) and a solution ofsodium periodate (84 mg) in water (1 ml) was added to the solution. Themixture was stirred for 30 minutes under ice-cooling and then the excessreagent was decomposed with sodium bisulfite. The resulting solution wasadjusted to pH 2 with 1N sodium hydroxide and concentrated to 5 ml underreduced pressure. The concentrate was passed through a column packedwith activated carbon (5 ml). The column was washed with water (30 ml)and eluted with a mixture of methanol and water. The eluate wasevaporated to dryness under reduced pressure. The residue was dissolvedin 50% aqueous methanol (10 ml) and the solution was stirred for 2.5hours at ambient temperature, maintaining the pH at 9.0 with 10% aqueoussodium carbonate. The solution was concentrated to about 5 ml. Theconcentrate was adjusted to pH 2.0 and passed through a column packedwith non-ionic adsorption resin, HP 20 (20 ml) and eluted with water.The eluate was lyophilized to giveD-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (4)(78 mg), whichwas identical with that of product prepared in Example 72-1.

EXAMPLE 111

(1) Step 1 ##STR451##

Heptanoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-PheOH(3) was prepared in substantially the same manner as step 1 of Example96 from compounds (1) and (2).

NMR (DMSO-d₆), δ(ppm): 0.7-2.4 (4.1H, m), 2.8-3.2 (2H, s), 3.4-4.8 (4H,m), 5.15 (2H, s), 6.7 (1H, m), 7.26 (5H, s), 7.40 (5H, s), 7.8 (1H, m),8.2 (2H, m), 8.7 (1H, m), 9.60 (1H, broad s).

(2) Step 2 ##STR452##

Heptanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-PheOH (4)was prepared in substantially the same manner as step 2 of Example 96.

NMR (CD₃ OD), δ(ppm): 0.91 (3H, m), 1.1-2.7 (20H, m), 1.46 (18H, s), 3.1(2H, m), 3.8-4.6 (4H, m), 7.19 (5H, s).

(3) Step 3 ##STR453##

Heptanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-PheOH (5) was prepared insubstantially the same manner as step 3 of Example 96.

NMR (D₂ O-NaOD), δ(ppm): 0.83 (3H, t, J=6 Hz), 1.0-2.1 (16H, m), 2.28(4H, t, J=7.5 Hz), 3.10 (2H, AB part of ABX system, J=14,8,4 Hz), 3.15(1H, t, J=7.5 Hz), 4.16 (2H, broad t, J=8 Hz), 4.50 (1H, dd, J=8,4 Hz),7.3 (5H, m).

EXAMPLE 112

(1) Step 1 ##STR454##

Heptanoyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-.gamma.-AbuOH(3) was prepared in substantially the same manner as step 1 of Example96 from compounds (1) and (2).

NMR (DMSO-d₆), δ(ppm): 0.7-2.6 (27H, m), 1.40 (18H, s), 2.8-4.6 (5H, m),5.11 (2H, s), 6.65 (1H, m), 7.34 (5H, s), 7.82 (2H, m), 8.16 (1H, d, J=7Hz), 8.60 (1H, m), 9.52 (1H, broad s).

(2) Step 2 ##STR455##

Heptanoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-.gamma.-AbuOH(4) was prepared in substantially the same manner as step 2 of Example96.

IR (Nujol): 1700(broad), 1640(broad), 1520(broad) cm⁻¹.

NMR (CD₃ OD), δ(ppm): 0.88 (3H, m), 1.1-2.6 (27H, m), 1.43 (18H, s),3.8-4.6 (3H, m).

(3) Step 3 ##STR456##

Heptanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-γ-AbnOH (5) was prepared insubstantially the same manner as step 3 of Example 96.

IR (Nujol): 1710, 1635 and 1540 cm⁻¹.

NMR (D₂ O), δ(ppm): 0.85 (3H, brt, J=6 Hz), 1.0-2.8 (24H, m), 3.28 (2H,t, J=6.5 Hz), 3.83 (1H, t, J=6 Hz), 4.1-4.5 (2H, m).

EXAMPLE 113

(1) Step 1 ##STR457##

Phenoxyacetyl-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-LeuOH(3) was prepared in substantially the same manner as step 1 of Example96 from compounds (1) and (2).

NMR (DMSO-d₆), δ(ppm): 0.7-2.4 (19H, m), 1.40 (18H, s), 3.6-4.7 (4H, m),4.55 (2H, s), 5.15 (2H, s), 6.5-8.8 (10H, m), 7.34 (5H, s), 9.55 (1H,broad s).

(2) Step 2 ##STR458##

Phenoxyacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-LeuOH(4) was prepared in substantially the same manner as step 2 of Example96.

NMR (CD₃ OD), δ(ppm): 0.90 (6H,broad d, J-5 Hz), 1.3-2.7 (13H, m), 1.43(18H, s), 3.8-5.0 (4H, m), 4.60 (2H, s), 6.8-7.6 (5H, m).

(3) Step 3 ##STR459##

Phenoxyacetyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-LeuOH (5) was prepared insubstantially the same manner as step 3 of Example 96.

NMR (CD₃ OD), δ(ppm): 0.88 (6H, m), 1.2-2.7 (13H, m), 3.73 (1H, brs),4.2-4.7 (3H, m), 4.55 (2H, s), 6.8-7.5 (5H, m).

EXAMPLE 114

(1) Step 1 ##STR460##

A suspension of D-TyrOH(oBzl)(596 mg) in a mixture ofbis(trimethylsilyl)acetamide (2 ml), dimethylformamide (2 ml) andmethylene chloride (2 ml) was stirred at ambient temperature for twohours. The resulting solution was added to a solution of the mixedanhydride ofD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc(1)(1.64 g) in a mixture of methylene chloride (10 ml) and ethyl acetate(5 ml). The reaction mixture was stirred at -15° C. for an hour and keptovernight in a refrigerator. The resulting solution was concentrated,taken up into ethyl acetate (50 ml) and washed in turn with dil.hydrochloric acid, water (X2) and brine. The resulting solution wasdried over magnesium sulfate and the solvent was distilled off to givean amorphous solid (2.11 g). The solid was triturated with ether andsupernatant was discarded to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-TyrOH(oBzl)(2)(1.74 g).

(2) Step 2 ##STR461##

1N Sodium hydroxide (18 ml) was added to a solution ofD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-TyrOH(oBzl)(2)(1.74 g) in a mixture of methanol (15 ml) and water (7 ml) andthe solution was stirred at 5° C. for 30 minutes and at ambienttemperature for 1.5 hours. The reaction mixture was neutralized to pH 7with 1N hydrochloric acid and concentrated in vacuo. The concentrate wasdiluted with water, acidified to pH 7 with 1N hydrochloric acid and thenextracted with ethyl acetate. The extract was washed with water andbrine, dried over magnesium sulfate and evaporated to giveD-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-D-TyrOH(oBzl)(3).

(3) Step 3 ##STR462##

D-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-TyrOH(oBzl)(4) wasprepared in substantially the same manner as step 3 of Example 96.

(4) Step 4 ##STR463##

A solution ofD-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-TyrOH(oBzl)(4)(258 mg)in a mixture of 0.25N hydrochloric acid (2 ml) and methanol (8 ml) washydrogenated over 10% palladium black (120 mg) for 21.5 hours. Thereaction mixture was filtered and the filtrate was evaporated todryness. The residue was dissolved in methanol (5 ml) and the solutionwas treated with 1N sodium hydroxide (1.5 ml) at 5° C. The resultingsolution was neutralized to pH 7, concentrated, adjusted to pH 2 andpassed through a column packed with HP 20 (16 ml). Elution was carriedout with aqueous methanol and then menanol. Fractions containing theobject compound were collected, evaporated, dissolved in water and thenlyophillized to giveD-Lac(OH)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-TyrOH (5)(100 mg).

NMR (D₂ O), γ(ppm): 1.0-2.5 (10H, m), 1.38 (3H, d, J=7 Hz), 1.41 (3H, d,J=7 Hz), 2.6-3.4 (2H, m), 3.74 (1H, t, J=6 Hz), 4.0-4.6 (5H, m), 6.82(2H, d, J=9 Hz), 7.10 (2H, d, J=9 Hz).

EXAMPLE 115

(1) Step 1 ##STR464##

Heptanoyl-β-D-Asp(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared in substantially the same manner as step 1 of Example 96 fromcompounds (10) and (2).

NMR (CDCl₃), δ(ppm): 0.7-1.0 (3H, m), 1.0-2.0 (32H, m), 2.0-2.5 (2H, m),2.8-3.1 (2H, m), 4.1-4.9 (2H, m), 4.9-5.1 (1H, m), 5.27 (2H, s), 5.5-6.0(1H, m), 7.0-7.3 (2H, m), 7.50 (5H, s), 8.9-9.2 (1H, m), 9.2-9.6 (1H,m).

(2) Step 2 ##STR465##

Heptanoyl-β-D-Asp(α-OH)-(L)-mesoDAP (4) was prepared in substantiallythe same manner as steps 1 and 2 of Example 96 from compound (3).

NMR (D₂ O), δ(ppm): 0.84 (3H, t, J=6 Hz), 1.2-2.0 (14H, m), 2.2-2.4 (2H,m), 2.5-3.0 (2H, m), 3.75 (1H, t, J=6 Hz), 4.0-4.3 (2H, m).

EXAMPLE 116

(1) Step 1 ##STR466##

Mandelyl(oAc)-δ-D-Aad(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared in substantially the same manner as step 1 of Example 96 fromcompounds (1) and (2).

NMR (CDCl₃), δ(ppm): 1.4 (18H, s), 1.2-2.0 (10H, m), 2.16 (3H, s),2.0-2.4 (2H, m), 4.0-4.2 (1H, m), 4.3-4.7 (2H, m), 5.08 (2H, s), 5.3-5.6(1H, m), 6.08 (1H, s), 6.7-7.1 (3H, m), 7.30 (10H, s), 8.8-9.0 (1H, m).

(2) Step 2 ##STR467##

Mandelyl-δ-D-Aad(α-OH)-(L)-mesoDAP (4) was prepared in substantially thesame manner as steps 2 and 3 of Example 96 from compound (3).

NMR (D₂ O), δ(ppm): 1.2-2.0 (10H, m), 2.1-2.4 (3H, m), 3.6-3.9 (1H, m),4.2-4.5 (2H, m), 5.17 (1H, s), 7.42 (5H, s).

EXAMPLE 117

(1) Step 1 ##STR468##

Heptanoyl-δ-D-Aad(α-oBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared in substantially the same manner as step 1 of Example 96 fromcompounds (1) and (2).

(2) Step 2 ##STR469##

Heptanoyl-δ-D-Aad(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) was preparedin substantially the same manner as step 2 of Example 96.

NMR (CD₃ OD): 0.7-1.1 (3H, m), 1.1-2.1 (36H, m), 2.1-2.6 (4H, m),3.9-4.6 (3H, m).

IR (Nujol): 3270, 1700, 1640, 1520, 1160 cm⁻¹.

(3) Step 3 ##STR470##

Heptanoyl-δ-D-Aad(α-OH)-(L)-mesoDAP (5) was prepared in substantiallythe same manner as step 3 of Example 96.

NMR (D₂ O), δ(ppm): 0.7-1.0 (3H, m), 1.1-2.1 (18H, m), 2.1-2.5 (4H, m),3.76 (1H, t, J=6 Hz), 4.2-4.4 (2H, m).

IR (Nujol): 3260, 1715, 1625, 1540 cm⁻¹.

EXAMPLE 118

(1) Step 1 ##STR471##

To a solution of L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(2)(561 mg) in the mixture of methylene chloride (20 ml) andbis(trimethylsilyl)acetamide (3.25 g) was added a solution of2-hexadesyl-3-acetoxyeicosanoyl chloride (1)(630 mg) in methylenechloride (5 ml).

The reaction mixture was left for 5 days at ambient temperature andconcentrated to give an oily residue to which water (20 ml) and ether(50 ml) were added. The organic layer was separated, dried overmagnesium sulfate and concentrated in vacuo. The residual paste wascolumn chromatographed on silicic acid and eluted with a mixture ofchloroform and methanol (2:1) to give a2-hexadesyl-3-acetoxyeicosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOE(3)(283 mg).

NMR (CDCl₃ +CD₃ OD), δ(ppm): 0.85 (6H, m), 1.94 (3H, s), 3.65-4.55 (m),5.02 (1H, m), 6.34 (1H, m), 6.98 (1H, m).

(2) Step 2 ##STR472##

2-Hexadesyl-3-acetoxyeicosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-OH(3)(200 mg) was dissolved in trifluoroacetic acid (2 ml) and the mixturewas left for 30 minutes at ambient temperature. The reaction mixture wasconcentrated and the residual oil was dissolved in n-butanol (15 ml) andthen 1N sodium hydroxide (1 ml) was added thereto. After standing for 2hours at ambient temperature, water (10 ml) and n-butanol (20 ml) wereadded to the reaction mixture. The n-butanol layer was separated andconcentrated in vacuo to give2-hexadesyl-3-hydroxyeicosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH(4)(150 mg.).

NMR (CD₃ OD), δ(ppm): 0.89 (6H, m), 3.65 (1H, m), 4.00 (1H, m),4.25-4.60 (4H, m).

EXAMPLE 119

(1) Step 1 ##STR473##

Pentaacetyl-D-Gluconyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH(2) was prepared in substantially the same manner as step 1 of Example1.

IR (Nujol): 3350-3300, 1730-1710, 1700-1690, 1650 cm⁻¹.

(2) Step 2 ##STR474##

Pentaacetyl-D-Gluconyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (3) wasprepared in substantially the same manner as step 2 of Example 17.

N.M.R. (CD₃ OD), δ(ppm): 1.37 (3H, d, J=7 Hz), 1.50-2.34 (10H, m), 2.00(9H, s), 2.10 (3H, s), 2.17 (3H, s), 3.92 (2H, s), 3.95-4.50 (6H, m),5.10-5.75 (4H, m).

(3) Step 3 ##STR475##

D-Gluconyl-L-Ala-γ-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (4) was prepared insubstantially the same manner as step 2 of Example 19.

N.M.R. (D₂ O), δ(ppm): 1.44 (3H, d, J=7 Hz), 1.34-2.50 (10H, m), 3.75(2H, s), 3.85 (2H, d, J=10 Hz), 3.84-4.67 (8H, m).

EXAMPLE 120

(1) Step 1 ##STR476##

5-(N-Z-5-oxo-4-oxazolidinyl)valeryl-L-Ala-γ-D-Glu-(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(2) was prepared in substantially the same manner as Step 1 of Example1.

N.M.R. (CD₃ OD), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.50 (18H, s), 1.38-2.50(18H, m), 4.00 (2H, s), 4.10-4.60 (5H, m), 4.27 (2H, s), 6.60 (2H, ABq,J=4 and 6 Hz), 7.43 (5H, s).

(2) Step 2 ##STR477##

α-Aminopimelyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(3) was prepared in substantially the same manner as step 2 of Example11.

N.M.R. (CD₃ OD), δ(ppm): 1.35 (3H, d, J=7 Hz), 1.42 (18H, s), 1.34-2.50(18H, m), 3.92 (2H, s), 3.63-4.50 (5H, m).

(3) Step 3 ##STR478##

α-Aminopimelyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (4) wasprepared in substantially the same manner as step 2 of Example 17.

N.M.R. (D₂ O), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.33-2.50 (18H, m), 3.73(1H, t, J=7 Hz), 3.83 (1H, t, J=7 Hz), 3.97 (2H, s), 4.07-4.30 (3H, m).

Preparation 85 ##STR479##

L-Ala-D-Glu(OH)OBzl (1) (942 mg) was suspended in methylene chloride (40ml), and bis(trimethylsilyl)acetamide (1.20 g) was added to thesuspension under stirring. The resulting solution was treated withstearoyl chloride (900 mg) at ambient temperature and left for an hour.Evaporation of the solvent gave an oily residue which was treated withwater to give white solids. The solids were collected by filtration,washed with water and dried over magnesium sulfate to givestearoyl-L-Ala-D-Glu(OH)OBzl (2) (1.60 g).

IR(Nujol): 3300, 2700-2500 (broad), 1720, 1710, 1660 (sh), 1640 cm⁻¹.

NMR(Pyridine-d₅): δ0.90 (3H, m), 1.0-3.0 (36H, m), 1.55 (3H, d, J=7 Hz),4.90-5.40 (2H, m), 5.23 (2H, s).

Preparation 86 ##STR480##

Stearoyl-L-Ala-D-Glu(OH)OBzl (1) (1.41 g) and N-hydroxysuccimide (280mg) were dissolved in tetrahydrofuran (50 ml). To the solution was addedN,N'-dicyclohexylcarbodiimide (500 mg) under ice-cooling and theresulting solution was left in a refrigerator overnight. The precipitateseparated were filtered and the filtrate was evaporated to give a whitesolid, which was recrystallized from a mixture of isopropylalcohol andisopropyl ether to give stearoyl-L-Ala-D-Glu(α-OBzl)OSu (2) (1.45 g).

IR(Nujol): 3300, 1805, 1780, 1740, 1660 (sh), 1640 cm⁻¹.

NMR(CDCl₃): δ0.87 (3H, m), 1.0-2.90 (39H, m), 2.80 (4H, s), 4.20-4.80(2H, m), 5.17 (2H, s), 6.20 (1H, d, 7 Hz), 7.37 (5H, s).

Preparation 87

2-Hexadecyloctadecanoyl-L-Ala-D-Glu(OH)OBzl was prepared insubstantially the same manner as that of Preparation 85.

IR(Nujol): 3300 (sh), 3280, 1740 (broad), 1640 (sh), 1625 cm⁻¹.

NMR(CDCl₃): δ0.83-2.67 (73H, m), 4.33-5.00 (2H, m), 5.15 (2H, s), 6.42(2H, d, J=8 Hz), 7.16 (5H, s).

Preparation 88

2-Hexadecyloctadecanoyl-L-Ala-D-Glu(α-OBzl)(γ-OSu) was prepared insubstantially the same manner as that of Preparation 86.

IR(Nujol): 3300, 1810, 1780, 1740, 1650 (sh), 1635 cm⁻¹.

NMR(CDCl₃): δ0.67-2.83 (73H, m), 2.80 (4H, s), 4.40-4.86 (2H, m), 5.16(2H, s), 6.60 (2H, d, J=7 Hz), 7.16 (5H, s).

Preparation 89

Stearoyl-D-Glu(OH)OBzl was prepared in substantially the same manner asthat of Preparation 85.

IR(Nujol): 3300, 1750, 1710, 1650 cm⁻¹.

NMR(CDCl₃): δ0.87 (3H, m), 1.0-2.70 (34H, m), 4.50-5.00 (1H, m), 5.17(2H, s), 6.33 (1H, d, J=8 Hz), 7.33 (5H, s), 10.30 (1H, s).

Preparation 90

Stearoyl-D-Glu(α-OBzl)Osu was prepared in substantially the same manneras that of Preparation 86.

IR(Nujol): 3300, 1810, 1780, 1740, 1650 cm⁻¹.

NMR(CDCl₃): δ0.87 (3H, m), 1.0-2.80 (36H, m), 2.76 (4H, s), 4.50-5.00(1H, m), 5.16 (2H, s), 6.32 (1H, d, J=8 Hz), 7.33 (5H, s).

Preparation 91

2-Hexadecyloctadecanoyl-D-Glu(OH)OBzl was prepared as substantially thesame manner as that of Preparation 85.

IR(Nujol): 3280, 1740, 1710, 1640 cm⁻¹.

NMR(CDCl₃): δ0.83-2.63 (70H, m), 4.33-5.00 (1H, m), 5.15 (2H, s), 6.20(1H, d, J=7 Hz), 7.30 (5H, s).

Preparation 92

2-Hexadecyloctadecanoyl-D-Glu(α-OBzl)Osu was prepared in substantiallythe same manner as that of Preparation 86.

IR(Nujol): 3300, 1810, 1780, 1745, 1640 cm⁻¹.

NMR(CDCl₃): δ0.83-2.83 (71H, m), 2.80 (4H, s), 4.50-5.00 (1H, m), 6.20(1H, d, J-7 Hz), 7.35 (5H, s).

Preparation 93

2-Docosyltetracosanoyl-L-Ala-D-Glu(OH)OBzl was prepared in substantiallythe same manner as that of Preparation 85.

IR(Nujol): 3270, 1740, 1640 (sh), 1625 cm⁻¹.

NMR(CDCl₃): δ0.67-2.50 (98H, m), 4.40-4.80 (2H, m), 5.10 (2H, s),6.16-6.40 (2H, m), 7.28 (5H, s).

Preparation 94

2-Docosyltetracosanoyl-L-Ala-D-Glu(α-OBzl)OSu was prepared insubstantially the same manner as that of Preparation 86.

IR(Nujol): 3300, 1815, 1780, 1750, 1650 (sh), 1640 cm⁻¹.

NMR(CDCl₃): δ0.67-3.00 (98H, m), 2.76 (4H, s), 4.40-4.83 (2H, m), 5.13(2H, s), 6.13 (2H, d, J=7 Hz), 7.30 (5H, s).

Preparation 95

2-Docosyltetracosanoyl-D-Glu(OH)OBzl was prepared in substantially thesame manner as Preparation 85.

IR(Nujol): 3250, 2600 (broad sh), 1740, 1710, 1645 cm⁻¹.

NMR(CDCl₃): 0.67-2.83 (95H, m), 4.50-5.00 (1H, m), 5.20 (2H, s), 6.20(1H, d, J=7 Hz), 7.36 (5H, s).

Preparation 96

2-Docosyltetracosanoyl-D-Glu(α-OBzl)OSu was prepared in substsantiallythe same manner as that of Preparation 86.

IR(Nujol): 3300, 1810, 1780, 1740, 1640 cm⁻¹. NMR(CDCl₃): δ0.68-2.87(96H, m), 2.87 (4H, s), 4.50-5.00 (1H, m), 5.20 (2H, s), 6.25 (1H, d,J=7 Hz), 7.36 (5H, s).

Preparation 97 ##STR481##

To a solution of Boc-(L)-Z-(D)-mesoDAP-(D)-NHNHBoc (1) (2.16 g) andN-methylmorpholine (404 mg) in methylene chloride (40 ml) was addedisobutylchloroformate (546 mg) at -10°--15° C. The mixture was stirredfor 30 minutes at ambient temperature.

A solution of D-AlaOEt, prepared from D-AlaOEt hydrochloric acid salt(614 mg) and N-methylmorphorin (404 mg) by stirring 10 minutes inmethylene chloride at ambient temperature, was added to the mixture. Theresulting mixture was stirred for 1.5 hours at -10°--15° C. Methylenechloride was evaporated in vacuo and the residue was dissolved in amixture of ethyl acetate (100 ml) and 2.5% hydrochloric acid (50 ml).The organic layer was separated and washed with water (50 ml) and 2%sodium bicarbonate (50 ml), dried over magnesium sulfate and then thesolvent was evaporated in vacuo. The residue was crystallized from ether(30 ml) to give Boc-(L)-Z-(D)-mesoDAP-(L)-D-AlaOEt-(D)-NHNHBoc (2) (2.2g).

IR (Nujol): 3300, 3230, 1720, 1705, 1675, 1645, 1630 cm⁻¹.

NMR(CD₃ OD), δ: 1.45 (18H, s), 1.2-2.0 (12H, m), 4.0-4.6 (5H, m), 5.13(2H, s), 7.37 (10H, s).

Preparation 98 ##STR482##

Boc-(L)-Z-(D)-mesoDAP-(L)-GlyOEt-(D)-NHNHBoc (2) was prepared insubstantially the same manner as that of Preparation 97.

IR (Nujol): 3270, 1670 (broad), 1240, 1160, 1040, 1020, 860 cm⁻¹.

NMR(CD₃ OD), δ: 1.28 (3H, t, J=7 Hz), 1.48 (18H, s), 1.4-2.0 (6H, m),3.98 (2H, s), 4.20 (2H, q, J=7 Hz), 3.9-4.2 (1H, m), 5.10 (2H, s), 7.35(5H, s).

Preparation 99 ##STR483##

Boc-(L)-Z-(D)-mesoDAP-(L)-GlyOMe-(D)-NHNHBoc (2) was prepared insubstantially the same manner as that of Preparation 97.

IR(Nujol): 3300, 3230, 1730, 1710, 1680, 1650, 1630 cm⁻¹.

NMR(CD₃ OD), δ: 1.47 (18H, s), 1.4-2.0 (6H, s), 3.68 (3H, s), 3.94 (2H,s), 3.9-4.3 (2H, m), 5.04 (2H, s), 7.30 (5H, s).

Preparation 100 ##STR484##

Boc-(L)-Z-(D)-mesoDAP-(L)-D-AlaOMe-(D)-NHNHBoc (2) was prepared insubstantially the same manner as that of Preparation 97.

IR(Nujol): 3300, 3230, 1725, 1705, 1680, 1645, 1630 cm⁻¹.

NMR(CD₃ OD), δ: 1.33 (3H, d, J=7 Hz), 1.48 (18H, s), 1.4-2.1 (6H, m),3.67 (3H, s), 3.8-4.3 (2 H, m), 4.35 (1H, q, J=7 Hz), 5.02 (2H, s), 7.30(5H, s).

Preparation 101 ##STR485##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOEt-(D)-NHNHBoc (2) was prepared insubstantially the same manner as that of Preparation 97.

IR(Nujol): 3300, 3220 (schoulder), 1735, 1710, 1680, 1645, 1630 cm⁻¹.

NMR(CD₃ OD), δ: 1.23 (3H, t, J=7 Hz), 1.37 (2H, d, J=7 Hz), 1.40 (9H,s), 1.4-2.0 (6H, m), 3.9-4.6 (5H, m), 5.10 (2H, s), 7.33 (5H, s).

Preparation 102 ##STR486##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOEt-(D)-NHNHBoc (1) (0.5 g) was added totrifluoroacetic acid (5 ml) and the mixture was stirred for 30 minutesat ambient temperature. After evaporation of trifluoroacetic acid, theresidue was dissolved in ethanol (10 ml) and the solution was cooled to0° C.

To the solution was added N-bromosuccinimide (420 mg) and the mixturewas stirred for 10 minutes at 0° C. Ethanol was evaporated in vacuo andthe residue was dissolved in water (10 ml). The solution was cooled to0° C. and the excess reagent was decomposed with aqueous sodiumbisufite. The mixture was neutralized to pH 8.0 with sodium bicarbonateand extracted with ethyl acetate (30 ml).

The extract was dried over magnesium sulfate and 2N hydrochloric acid inethyl acetate (4 ml) was added to the extract. The solvent wasevaporated in vacuo and the residue was crystallized from ether to givehydrochloric acid salt of Z-(L)-mesoDAP-(L)-D-(AlaOEt-(D)-OEt (2) (280mg).

IR(Nujol): 3280, 1730, 1690, 1650 cm⁻¹.

NMR (CD₃ OD), δ: 1.23 (3H, t, J=7 Hz, 1.30 (3H, t, J=7 Hz), 1.35 (3H, d,J=7 Hz), 1.5-2.1 (6H, m), 3.9-4.6 (7H, m), 5.10 (2H, s), 7.32 (5H, s).

Preparation 103 ##STR487##

Z-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe hydrochloric acid salt (2) was preparedin substantially the same manner as that of Preparation 102.

IR(CHCl₃): 1745, 1720, 1680 cm⁻¹.

Preparation 104 ##STR488##

Z-(D)-mesoDAP-(L)-D-AlaOEt-(D)-OEt hydrochloric acid salt (2) wasprepared in substantially the same manner as that of Preparation 102.

IR(Nujol): 3300, 1725, 1680, 1655 cm⁻¹.

NMR(CD₃ OD), δ: 1.1-2.0 (15H, m), 3.9-4.5 (7H, m), 5.08 (2H, s), 7.32(5H, s).

Preparation 105 ##STR489##

Z-(D)-mesoDAP-(L)-D-AlaOMe-(D)-OMe hydrochloric acid salt (2) wasprepared in substantially the same manner as that of Preparation 102.

NMR(CD₃ CD), δ: 1.42 (3H, d, J=7 Hz), 1.4-2.0 (6H, m), 3.72 (6H, s),3.95 (1H, t, J=6 Hz), 4.23 (1H, t, J=6 Hz), 4.47 (1H, q, J=7 Hz), 5.10(1H, s), 7.38 (5H, s).

Preparation 106 ##STR490##

To a solution of bis(trimethylsilyl)acetamide (15 ml) and methylenechloride (60 ml) was added Boc-(L)-mesoDAP-(D)-NHNHBoc (1) (6.08 g). Themixture was stirred for an hour at ambient temperature and cooled to 0°C. and carbobenzyloxy chloride (5.1 g) was added thereto. The resultingmixture was stirred for an hour at ambient temperature. Afterevaporation of methylene chloride, the residue was poured into a mixtureof ethyl acetate (60 ml) and 2.5% hydrochloric acid (50 ml). The organiclayer was separated and washed with aqueous sodium chloride, dried overmagnesium sulfate and then evaporated. The residue was pulverized withisopropyl ether to give Boc-(L)-Z-(D)-mesoDAP-(D)-NHNHBoc (2) (5.4 g).

NMR(CD₃ OD), δ: 1.43 (18H, s), 1.42-2.0 (6H, m), 3.9-4.3 (2H, m), 5.08(2H, s), 7.32 (5H, s).

IR(Nujol): 3270, 3100-2200, 1690, 1370, 1240, 1160, 1050, 1020 cm⁻¹.

Preparation 107 ##STR491##

DiZ-mesoDAP-(L)-AlaOEt-(D)-OEt (2) was prepared in substantially thesame manner as that of Preparation 106.

[α]_(D) =+16.3° (C=1, MeOH).

IR(Nujol): 3300, 1740 (schoulder), 1730, 1685, 1650 cm⁻¹.

NMR(CD₃ OD), δ: 1.20 (6H, t, J=7 Hz), 1.37 (3H, d, J=7 Hz), 1.4-2.0 (6H,m), 4.0-4.5 (7H, m), 5.10 (4H, s), 7.33 (10H, s).

Preparation 108 ##STR492##

DiZ-mesoDAP-(L)-D-AlaOEt-(D)-OEt (2) was prepared in substantially thesame manner as that of Preparation 106. This compound was identifiedwith the product prepared in Preparation 107.

Preparation 109 ##STR493##

Z-(L)-Boc-(D)-mesoDAP (2) was prepared in substantially the same manneras that of Preparation 106.

IR(Nujol): 3300, 2600 (broad, sh), 1750-1650 (broad) cm⁻¹.

NMR(DMSO-d₆), δ: 1.20-2.0 (15H, m), 3.63-4.16 (2H, m), 5.06 (2H, s),6.93 (1H, d, J=8 Hz), 7.33 (5H, s).

Preparation 110 ##STR494##

O-acetylbenziloyl-D-Glu(OH)OBzl (2) was prepared in substantially thesame manner as that of Preparation 85.

NMR(CDCl₃), δ: 2.16 (3H, s), 1.9-2.5 (4H, m), 4.5-4.9 (1H, m), 5.16 (2H,s), 7.03 (1H, d, J=8 Hz), 7.1-7.7 (15H, m), 9.55 (1H, s).

IR(film): 1730, 1680, 1510, 1210, 1025, 750, 695 cm⁻¹.

Preparation 111 ##STR495##

Diphenylacetyl-D-Glu(OH)OBzl (2) was prepared in substantially the samemanner as that of Preparation 85.

NMR(CDCl₃), δ: 1.7-2.5 (4H, m), 4.5-4.9 (1H, m), 4.95 (1H, s), 5.10 (2H,s), 6.37 (1H, d, J=8 Hz), 7.23 (10H, s), 7.30 (5H, s), 9.40 (1H, s).

IR(CHCl₃): 3400, 1730, 1710, 1670, 1490 cm⁻¹.

Preparation 112 ##STR496##

Cinnamoyl-D-Glu(OH)OBzl (2) was prepared in substantially the samemanner as that of Preparation 85.

NMR(CDCl₃), δ: 1.9-2.7 (4H, m), 4.7-5.0 (1H, m), 5.17 (2H, s), 6.43 (1H,d, J=16 Hz), 6.74 (1H, d, J=8 Hz), 7.30 (10H, s), 7.63 (1H, d, J=16 Hz),10.07 (1H, s).

IR(Nujol): 3300, 1735, 1690, 1650, 1620, 1525, 1280, 1205, 975, 750, 695cm⁻¹.

Preparation 113 ##STR497##

Adamantane-1-carbonyl-D-Glu(OH)OBzl (2) was prepared in substantiallythe same manner as that of Preparation 85.

NMR(CDCl₃), δ: 1.5-2.6 (19H, m), 4.5-4.9 (1H, m), 5.16 (2H, s), 6.52(1H, d, J=8 Hz), 7.33 (5H, s), 9.80 (1H, s).

IR(film): 4300, 1730, 1710, 1630, 1520, 1210 cm-1.

Preparation 114 ##STR498##

Z-(L)-Boc(D)-mesoDAP (1) was dissolved in acetic acid (50 ml) andhydrogenated over palladium black (450 mg) at ambient temperature. Thecatalyst was removed by filtration and the filtrate was evaporated invacuo to give a white solid. The solid was dissolved in water (100 ml)and chromatographed on a column of macroporous non-ionic adsorptionresin, Hp 20 (200 ml). The column was eluted successively with water and50% aqueous methanol. The latter fractions were combined and evaporatedto give Boc-(D)-mesoDAP (2) (3.80 g).

IR(Nujol): 3400, 2600 (broad), 1710, 1640, 1580 cm⁻¹.

NMR(D₂ O): δ1.16-2.16 (15H, m), 3.60-4.20 (2H, m).

EXAMPLE 121

(1) Step 1 ##STR499##

L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (1.64 g) (1) wasdissolved in a mixture of dioxane (30 ml) and water (30 ml). To thissolution, triethylamine (606 mg) was added. To the mixture was added10-(2,3-dimethoxy-5-methyl-1,4-benzoquino-6-yl) decanoic acidN-hydroxy-succinimide ester (872 mg). The reaction mixture was kept atroom temperature for 24 hours. The reaction mixture was concentrated andextracted with ethylacetate. The organic layer was washed successivelywith aqueous hydrochloric acid, water and brine, and dried overmagnesium sulfate. Evaporation of the solvent gave a foam, which waspulvirized with ether to afford10-(2,3-dimethoxy-5-methyl-1,4-benzoquino-6-yl)decanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-AlaOH(2) (0.97 g).

IR(Nujol): 3280, 1710, 1640, 1610 cm⁻¹.

NMR (CDCl₃), δ: 1.48 (9H, s), 2.00 (3H, s), 4.00 (6H, s), 3.66-4.66 (5H,m).

(2) Step 2 ##STR500##

10-(2,3-dimethoxy-5-methyl-1,4-benzoquino-6-yl)decanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(2) (0.945 g) was dissolved in trifluoroacetic acid (20 ml). Thesolution was kept for 1 hour at room temperature. The reaction mixturewas concentrated in vacuo. The residue was dissolved in water. The pH ofthe aqueous solution was adjusted to 2-3, and the whole solution wassubjected to Hp-20 column (100 ml). The column was eluted with a mixtureof water and MeOH-H₂ O (1:2). Evaporation of the latter fractions gavean orange foam, which was dissolved in water and lyophillized to give10-(2,3-dimethoxy-5-methyl-1,4-benzoquino-6-yl)-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-D-AlaOH(3) (0.80 g).

IR(Nujol): 3300, 1720, 1640, 1610, 1530 cm⁻¹.

NMR(CD₃ CD), δ: 2.00 (3H, s), 3,92 (6H, s) 3.60-4.50 (5H, m).

EXAMPLE 122

(1) Step 1 ##STR501## 10-(2,3,4-Trimethoxy-6-methylphenyl)-decanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-AlaOH (2)was prepared in substantially the same manner as that of Step 1 ofExample 121.

IR(Nujol): 1710, 1640, 1530 cm⁻¹.

NMR(CDCl₃): δ1.46 (9H, s), 2.27 (3H, s), 3.90 (9H, s), 4.00-5.00 (5H, m)6.50 (1H, s).

(2) Step 2 ##STR502##

10-(2,3,4-Trimethoxy-6-methylphenyl)-decanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH(3) was prepared in substantially the same manner as that of step 2 ofExample 121.

IR(Nujol): 3260, 1720, 1620, 1530 cm⁻¹.

NMR(CD₃ OD): δ2.26 (3H, s), 3.83 (9H, s), 6.60 (1H, s).

EXAMPLE 123

(1) Step 1 ##STR503##

O-Stearoyl-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (2)was prepared in substantially the same manner as step 1 of Example 121.

IR(Nujol): 3300, 1720, 1630 cm⁻ 1.

NMR(CDCl₃), δ: 0.90 (3H, m), 4.15-4.90 (5H, m), 5.17 (1H, m).

(2) Step 2 ##STR504##

O-Stearoyl-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (3) wasprepared in substantially the same manner as step 2 of Example 121.

IR(Nujol): 3370, 1740, 1650 (sh), 1630 cm⁻¹.

NMR(D₂ O-NaHCO₃), δ: 0.87 (3H, m), 3.74 (1H, m), 4.0-4.5 (4H, m), 5.10(1H, m), [α]_(D) =-38.24° (C=0.30, 5% NaHCO₃).

EXAMPLE 124

(1) Step 1 ##STR505##

n-Tetracosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (2)was prepared in substantially the same manner as that of step 1 ofExample 121.

IR(Nujol): 3300, 1750, 1630 cm⁻¹.

NMR(CDCl₃), δ: 0.89 (3H, m), 4.0-4.9 (5H, m).

(2) Step 2 ##STR506##

n-Tetracosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (3) wasprepared in substantially the same manner as that of step 2 of Example121.

IR(Nujol): 3280, 1730, 1630 cm⁻¹.

NMR(D₂ O+NaHCO₃), δ: 0.88 (3H, m), 3.72 (1H, m), 3.95-4.6 (4H, m).

EXAMPLE 125

(1) Step 1 ##STR507##

(D)-Boc-mesoDAP (1) (580 mg) was dissolved in a mixture of methanol (10ml), methylene chloride (40 ml) and triethylamine (404 mg). To thissolution was added stearoyl-L-Ala-D-Glu(α-OBzl)OSu (1.34 g). Theresulting solution was left overnight at ambient temperature, evaporatedand extracted with ethyl acetate after acidification. The organic layerwas washed with water, dried over magnesium sulfate and evaporated togive stearoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(3) (1.10 g).

IR(Nujol): 3300, 2700-2500 (broad), 1720 (broad), 1650 (sh), 1630 cm⁻¹.

NMR(DMSO-d₆): δ0.88 (3H, m), 1.0-2.50 (54H, m), 4.0-4.50 (4H, m), 5.12(2H, s), 7.36 (5H, s), 7.84 (1H, d, J=7 Hz), 8.04 (1H, d, J=7 Hz), 8.28(1H, d, J=7 Hz).

(2) Step 2 ##STR508##

Stearoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-DAP (3) (1.10 g) wasdissolved in acetic acid (80 ml) and hydrogenated over 10% palladiumblack (120 mg). The catalyst was removed by filtration and the filtratewas evaporated to give a gummyresidue which was dissolved intrifluoroacetic acid (5 ml). After standing for 15 minutes at ambienttemperature, the solvent was evaporated to give a foam which wastriturated with diethyl ether to givestearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP (4) (0.90 g).

IR(Nujol): 3300, 2600-2300, 1720, 1650 (sh), 1630 cm⁻¹.

NMR(NaOD-D₂ O): δ0.84 (3H, m), 1.0-2.60 (45H, m), 3.10-3.40 (1H, m),4.0-4.50 (3H, m).

EXAMPLE 126

(1) Step 1 ##STR509##

Stearoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP (3) was prepared insubstantially the same manner as step 1 of Example 125.

IR(Nujol): 3300, 2600-2300 (broad), 1750-1700 (broad), 1650 (broad)cm⁻¹.

NMR (DMSO-d₆): δ0.86 (3H, m), 1.0-2.40 (5H, m), 4.0-4.50 (3H, m), 5.13(2H, s), 6.90 (1H, m), 7.36 (5H, s), 7.70-8.30 (2H, m).

(2) Step 2 ##STR510##

Stearoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (4) was prepared in substantially thesame manner that of step 2 of Example 125.

IR(Nujol): 3250 (sh), 1720, 1640 cm⁻¹.

NMR(NaOD-D₂₆): δ0.80-2.80 (45H, m), 3.12-3.40 (1H, m), 4.0-4.32 (2H, m).

EXAMPLE 127

(1) Step 1 ##STR511##

2-Hexadecyloctadecanoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP (3) wasprepared in substantially the same manner as that of step 1 of Example125.

IR(Nujol): 3300, 1730 (broad), 1680, 1640 cm⁻¹.

NMR(CDCl₃): δ0.87-2.87 (77H, m), 4.33-4.80 (3H, m), 5.16 (2H, s), 7.33(5H, s).

(2) Step 1 ##STR512##

2-Hexadecyloctadecanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (4) was prepared insubstantially the same manner as that of step 2 of Example 125.

IR(Nujol): 3250, 1720, 1630 cm⁻¹.

NMR(CDCl₃): δ0.68-2.68 (77H, m), 3.68-4.00 (1H, m), 4.16-4.83 (2H, m).

EXAMPLE 128

(1) Step 1 ##STR513##2-Hexadecyloctadecanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(3) was prepared in substantially the same manner as that of step 1 ofExample 125.

IR(Nujol): 3300, 1745, 1660 (sh), 1640 cm⁻¹.

(2) Step 2 ##STR514##

2-Hexadecyloctadecanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (4)was prepared in substantially the same manner as that of step 2 ofExample 125.

IR(Nujol): 3270, 1730, 1630 cm⁻¹.

NMR(CF₃ CO₂ H): δ0.92 (6H, m) 4.30-5.00 (5H, m).

EXAMPLE 129

(1) Step 1 ##STR515##

2-Docosyltetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(3) was prepared in substantially the same manner as that of step 1 ofExample 125.

IR(Nujol): 3250, 1750 (sh), 1715 (sh), 1690, 1660, 1640 cm⁻¹.

(2) Step 2 ##STR516##

2-Docosyltetracosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (4)was prepared in substantially the same manner as that of step 2 ofExample 125.

IR(Nujol): 3300, 1720, 1645-1625 (broad) cm⁻¹.

NMR(CDCl₃): δ0.67-3.00 (107H, m), 4.33-4.67 (3H, m).

EXAMPLE 130

(1) Step 1 ##STR517##

2-Docosyltetracosanoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP (3) wasprepared in substantially the same manner as that of step 1 of Example125.

IR(Nujol): 3300, 1740 (sh), 1720 (broad), 1690, 1640 cm⁻¹.

NMR(CDCl₃): δ0.86-2.50 (101H, m), 4.10-4.83 (3H, m), 5.13 (2H, s), 7.30(5H, s).

(2) Step 2 ##STR518##

2-Docosyltetracosanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (4) was prepared insubstantially the same manner as that of step 2 of Example 125.

IR(Nujol): 3300, 1720, 1640-1620 (broad) cm⁻¹.

NMR(CDCl₃): δ0.68-2.86 (101H, m), 4.33-4.86 (2H, m).

EXAMPLE 131

(1) Step 1 ##STR519##

2-Docosyltetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(1) (1.50 g) was dissolved in a mixture of tetrahydrofuran (50 ml) andmethylene dichloride (20 ml). To this solution was added etheralsolution (6 ml) of diazomethane (0.5 Mole solution) under stirring andreacted for 1 hour. The excess of diazomethane was destroyed by addingacetic acid and the resulting solution was evaporated to give a waxyresidue, which was subjected to a silica gel column. Elution withchloroform-methanol (20:1) and subsequent evaporation gave2-docosyltetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(OMe)-(D)-OMe(2) (0.90 g).

IR(Nujol): 3300, 1755 (sh), 1745, 1690, 1660, 1640 cm⁻¹.

NMR(CDCl₃): δ0.86-2.50 (116H, m), 3.68 (3H, m), 3.72 (3H, s), 4.16-4.83(5H, m), 5.15 (2H, s), 7.33 (5H, s).

(2) Step 2 ##STR520##

2-Docosyltetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(OMe)-(D)-OMe(3) (1.0 g) was dissolved in acetic acid (40 ml) and then treated withhydrogen chloride saturated acetic acid (2 ml). After standing for 2hours at ambient temperature, the solution was evaporated to give awhite residue, which was throughly washed with diisopropylether toafford2-docosyltetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-mesoDAP-(L)-D-Ala(OMe)-(D)-OMe(4) (0.90 g).

IR(Nujol): 3300, 1750, 1660, 1640 cm⁻¹.

NMR(CDCl₃): δ0.68-3.00 (107H, m), 3.68-4.00 (6H, broad), 4.33-5.86 (4H,m), 5.16 (2H, s), 7.36 (5H, s).

EXAMPLE 132

(1) Step 1 ##STR521##

To a solution of Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc (2) (2.76 g)and triethylamine in aceton-water (2:1, 90 ml) was added a solution ofO-acetylbenziloyl-γ-D-Glu(α-OBzl)-1-succinimidyl ester (3.4 g:5.8 mM) inacetone at 0° C. with stirring. Stirring was continued overnight,allowing the temperature of the mixture to reach to room temperature.After evapolation of acetone, the residue was added ethyl acetate (60ml), and the organic layer was washed with dil sodium bicarbonate,saturated sodium chloride, dil hydrochloric acid and water. After dryingover magnesium sulfate, the solvent was removed in vacuo and trituratedwith isopropyl ether to giveO-acetylbenziloyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(3) (4.63 g).

NMR(CDCl₃): δ1.0-2.0 (29H, m), 2.05 (3H, s), 2.0-2.6 (2H, m), 4.0-5.0(4H, m), 5.13 (2H, s), 5.66 (1H, broads), 6.80 (3H, m), 7.07 (2H, m),7.33 (15H, s), 8.94 (1H, s).

IR(Nujol): 3300, 1735, 1670, 1520, 1240, 1165, 700 cm⁻¹.

(2) Step 2 ##STR522##

To a solution ofO-acetylbenziloyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(3) (4.2 g) in methanol (25 ml) was added 1N sodium hydroxide (16 ml) at0° C. with stirring. The mixture was stirred in an ice bath for 30minutes and then at room temperature for 30 minutes. After addition of1N hydrochloric acid (16 ml), the solvent was removed in vacuo, and theresidue was dissolved in ethyl acetate (60 ml) and water (40 ml). Theorganic layer was washed with saturated sodium chloride and dried overmagnesium sulfate and evaporated and then triturated with diethyl etherto givebenziloyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc (4)(2.97 g).

NMR (CD₃ OD): δ1.23 (3H, d, 7 Hz), 1.0-2.0 (26H, m), 2.0-2.6 (2H, m),3.9-4.7 (4H, m), 7.1-7.7 (10H, m).

IR(Nujol): 3300, 1720, 1650, 1510, 1240, 1160, 700 cm⁻¹.

(3) Step 3 ##STR523##

Benziloyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc (4)(2.5 g) was treated with trifluoroacetic acid (10 ml) for 15 minutes atroom temperature. Resulting trifluoroacetic acid salt ofbenziloyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH-(D)-NHNH₂ (2.44 g) wasdissolved in 1N sulfuric acid (18 ml) and added sodium metaperiodate(0.62 g) at 0° C with stirring. After stirring for 20 minutes at 0° C.,the reaction mixture was treated with sodium bisulfite, and put on acolumn of Hp-20 (170 ml). After removal of inorganic salts with water,elution was carried out with methanol-water (1:1). The eluate waslyophilized to give benziloyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5)(1.0 g) [α]_(D) ²¹ =+11.90 (CO.36 H₂ O).

NMR(D₂ O): 1.36 (3H, d, 7 Hz), 1.3-2.1 (8H, m), 2.1-2.5 (2H, m), 3.76(1H, t, 6 Hz), 4.0-4.6 (3H, m), 7.45 (10H, d, 3 Hz).

IR(Nujol): 3300, 1710, 1645, 1520, 1220, 700 cm⁻¹.

EXAMPLE 133

(1) Step 1 ##STR524##

Diphenylacetyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(3) was prepared in substantially the same manner as that of step 1 ofExample 132.

NMR(DMSO-d₆): δ1.0-2.3 (31H, m), 3.7-4.5 (4H, m), 5.0 (1H, s), 5.06 (2H,s), 6.3-7.0 (1H, m), 7.23 (10H, s), 7.28 (5H, s), 7.5-8.1 (3H, m),7.3-9.0 (2H, m), 9.5 (1H, s).

IR(Nujol): 3300, 1725, 1630, 1525, 1220, 1160, 700 cm⁻¹.

(2) Step 2 ##STR525##

Diphenylacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(4) was prepared in substantially the same manner as that of step 2 ofExample 132.

NMR(CD₃ OD): δ1.36 (3H, d, J=7 Hz), 1.3-2.5 (28H, m), 3.9-4.6 (4H, m),5.10 (1H, s), 7.30 (10H, d, J=3 Hz).

IR(Nujol): 3300, 1720, 1650, 1520, 1220, 1160, 700 cm⁻¹.

(3) Step 3 ##STR526##

Diphenylacetyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was prepared insubstantially the same manner as that of step 3 of Example 132. [α]_(D)²² =+4.02 (C 0.2 H₂ O)

NMR(D₂ O): δ1.35 (3H, d, J=7 Hz), 1.3-2.6 (10H, m), 3.75 (1H, t, J=6Hz), 4.0-4.6 (3H, m), 5.20 (1H, s), 7.36 (10H, s).

IR(Nujol): 3250, 1720, 1620, 1525, 1220, 700 cm⁻¹.

EXAMPLE 134

(1) Step 1 ##STR527##

Cinnamoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(3) was prepared in substantially the same manner as that of step 1 ofExample 132.

NMR(CD₃ OD): δ1.1-2.6 (31H, m), 3.9-4.7 (4H, m), 5.19 (2H, s), 6.67 (1H,d, J=16 Hz), 7.1-7.9 (1H, m).

IR(Nujol): 3260, 1730, 1650, 1625, 1520, 1160 cm⁻¹.

(2) Step 2 ##STR528##

Cinnamoyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc (4)was prepared in substantially the same manner as that of step 2 ofExample 132

NMR(CD₃ OD): δ1.1-2.6 (31H, m), 3.9-4.7 (4H, m), 6.67 (1H, d, J=16 Hz),7.3-7.9 (6H, m).

IR(Nujol): 3260, 1740, 1690, 1655, 1620, 1550, 1160 cm⁻¹.

(3) Step 3 ##STR529##

Cinnamoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was prepared insubstantially the same manner as that of step 3 of Example 132.

[α]_(D) ²² =+5.69° (C 0.22H₂ O).

NMR(D₂ O): δ1.38 (3H, d, J=7 Hz), 1.5-2.6 (10H, m), 3.74 (1H, t, J=6Hz), 4.1-4.6 (3H, m), 6.65 (1H, d, J=16 Hz), 7.2-7.7 (6H, m).

IR(Nujol): 3250, 1720, 1650, 1620, 1530, 1220 cm⁻¹.

EXAMPLE 135

(1) Step 1 ##STR530##

Adamantane-1-carbonyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(3) was prepared in substantially the same manner as that of step 1 ofExample 132.

NMR(DMSO-d₆): δ1.0-2.3 (46H, m), 4.0-4.5 (4H, m), 5.10 (2H, s), 6.5-6.9(1H, m), 7.33 (5H, s), 7.5-8.6 (5H, m), 9.53 (1H, s).

IR(Nujol): 3300, 1740 (sholder), 1720, 1685, 1630, 1520, 1240, 1160, 700cm⁻¹.

(2) Step 2 ##STR531##

Adamantane-1-carbonyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-NHNHBoc(4) was prepared in substantially the same manner as that of step 2 ofExample 132.

NMR(CD₃ OD): δ1.3-2.5 (46H, m), 3.9-4.6 (4H, m).

IR(Nujol): 3300, 1720, 1640, 1520, 1240, 1160 cm⁻¹.

(3) Step 3 ##STR532##

Adamantane-1-carbonyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) wasprepared in substantially the same manner as that of step 3 of Example132.

[α]_(D) ²² =-2.64 (C 0.23 H₂ O).

NMR(D₂ O): δ1.40 (3H, d, J=7 Hz), 1.2-2.6 (25H, m), 3.80 (1H, t, J=6Hz), 4.2-4.5 (3H, m).

IR(Nujol): 3250, 1720, 1635, 1530 cm⁻¹.

EXAMPLE 136

(1) Step 1 ##STR533##

To a solution of D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (1)(1.2 g) in water (5 ml) was added triethylamine (0.96 g) and a solutionof benzyloxycarbonyloxyimino-2-phenylacetonitrile (0.69 g) in acetone (5ml) at 0° C. with stirring and the mixture was stirring over night atroom temperature. After evapolation of acetone to the residue was addedwater (5 ml) and ethyl acetate (5 ml). The aqueous layer was separated,washed with ethyl acetate (5 ml), acidified with 1N-hydrochloric acid,and extracted with ethyl acetate (20 ml). The extract was washed withwater (5 ml×2) and dried over magnesium sulfate. Evaporation of thesolvent gave 1.42 g (94.7%) ofD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Z-(D)-mesoDAP-(L)-GlyOH (2).

(2) Step 2 ##STR534##

To a solution of D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Z-(D)-mesoDAP-(L)-GlyOH(2) (1.42 g) in methanol (30 ml) was added a solution of diazomethane indiethyl ether at 0° C. with stirring. The mixture was stirred for 4hours at 0° C. and for 2 hours at room temperature and allowed to standovernight. After addition of acetic acid (0.1 ml) the solvent wasevapolated in vacuo and crystallized from ethyl acetate to giveD-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-Z-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe (3)(1.30 g).

NMR(CD₃ OD): δ1.33 (3H, d, J=7 Hz), 1.36 (3H, d, J=7 Hz), 1.5-2.5 (10H,m) 3.67 (9H, s), 3.95 (2H, s), 4.0-4.6 (4H, m), 5.06 (2H, s), 7.47 (5H,s).

IR(Nujol): 3310, 1760, 1740, 1690, 1660, 1630, 1550, 1280 cm⁻¹.

(3) Step 3 ##STR535##

A solution ofD-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-Z-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe (3) in80% methanol (30 ml) was hydrogenolyzed in the presence of 10% palladiumcharcoal (0.12 g). After the catalyst was removed, the solvent wasevapolated in vacuo and crystallized from methanol-diethylether to giveD-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-GlyOMe-(D)-OMe (4) (0.86 g).

NMR(D₂ O): δ1.43 (3H, d, J=7 Hz), 1.53 (3H, d, J=7 Hz), 1.5-2.6 (10H,m), 3.6-3.8 (1H, m), 3.77 (9H, s), 4.03 (2H, s), 4.1-4.6 (4H, m).

IR(Nujol): 3370, 1730, 1630, 1520, 1210 cm⁻¹.

EXAMPLE 137 ##STR536##

A solution of Z-(L)-mesoDAP-(L)-GlyOH (2) (186 mg) in water (3 ml) wasadjusted to pH 8 with triethylamine and a solution ofD-Lac(oAc)-L-Ala-D-Glu(oSu) (α-oBzl) (1) (260 mg) in dioxane (2 ml) wasadded thereto.

The mixture was stirred at ambient temperature for five hours,maintaining the pH 8 with triethylamine, and then evaporated.

The aqueous layer was diluted with water (10 ml) and adjusted to pH 4with 1N hydrochloric acid. This solution was washed with ethyl acetateand adjusted to pH 2 with 1N hydrochloric acid and then extracted withethyl acetate. The extract was washed with a saturated sodium chloride,dried over magnesium sulfate and evaporated to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(D)-Z-(L)-mesoDAP-(L)-GlyOH (3) (203mg).

N.M.R. (CD₃ OD), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.45 (3H, d, J=7 Hz),1.4-2.0 (6H, m), 2.10 (3H, s), 2.0-2.4 (4H, m), 3.93 (2H, s), 4.1-4.7(4H, m), 4.9-5.1 (1H, m), 5.12 (2H, s), 5.20 (2H, s), 7.37 (10H, s).

EXAMPLE 138 ##STR537##

A solution ofD-Lac(oAc)-L-Ala-γ-D-Glu(α-OBzl)-(D)-Z-(L)-mesoDAP-(L)-GlyOH (1) (180mg) in methanol (30 ml) was hydrogenated over 10% palladium-black (50mg) under an ordinary atmospheric pressure of hydrogen. The reactionmixture was filtered and the filtrate was concentrated. The concentratewas dissolved in methanol (1 ml) and triturated with ether and thenfiltered. The powder obtained was washed with ether to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-OH)-(D)-mesoDAP-(L)-GlyOH (2) (98 mg).

N.M.R. (D₂ O), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.43 (3H, d, J=7 Hz),1.5-2.2 (10H, m), 2.0 (3H, s), 3.90 (2H, s), 4.0-4.5 (5H, m).

EXAMPLE 139 ##STR538##

A solution of D-Lac-(oAc)-L-Ala-γ-D-Glu(α-OH)-(D)-mesoDAP-(L)-GlyOH (1)(105 mg) in methanol (5 ml) was stirred at ambient temperature for threehours, maintaining the pH at 9 with 5% aqueous potassium carbonate.

The solution was evaporated and the residue was adjusted to pH 3.5 andput on a column of activated carbon. The column was washed with waterand eluted with 70% aqueous acetone. The eluate was evaporated and theresidue was dissolved in methanol (0.5 ml) and triturated with ether.The precipitate thus obtained was filtered and washed ether to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-(D)-mesoDAP-(L)-GlyOH (2) (60 mg).

N.M.R. (D₂ O), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.44 (3H, d, J=7 Hz),1.7-2.5 (10H, m), 3.98 (2H, s), 4.08 (1H, t, J=6 Hz), 4.2-4.5 (5H, m).

EXAMPLE 140 ##STR539##

To a cold mixture of D-Lac(oAc)-L-Ala-γ-D-Glu(OH) (α-oBzl) (1) (1.05 g)and N-hydroxysuccinimide (317 mg) in a mixture of dioxane (10 ml) andtetrahydrofuran (2 ml) was added N,N'-dicyclohexylcarbodiimide (557 mg).The mixture was stirred at 10° C. for ten minutes and at ambienttemperature overnight.

The precipitated N,N'-dicyclohexylurea was filtered off and washed withdioxane and then filtered. The combined filtrate was evaporated and theresidue was dissolved in dioxane (6 ml). To this solution was added asolution of Boc-(D)meso-DAP-(D)GlyOH (2) (600 mg) and N-methylmorpholine(0.57 ml) in dimethylformamide (6 ml).

The resulting solution was stirred at °C. for 30 minutes and thestirring was continued at ambient temperature for 2.3 hours and then anadditional N-methylmorpholine (0.10 ml) was added thereto. The mixturewas stirred at the same temperature for 2 hours. The reaction mixturewas concentrated, diluted with water and then washed with ether. Theaqueous layer was cooled, acid acidifed to pH 2 with 1N aqueoushydrochloric acid and then extracted four times with a mixture ofmethylene chloride and ethyl acetate (1:1). The combined extracts werewashed twice with water and brine, dried over anhydrous magnesiumsulfate and then evaporated to give amorphous solid. (1.2 g). The solidwas dissolved in methylene chloride (2 ml) and diluted with ether (30ml) to give an oily precipitate and the supernatant was decanted. Thisoperation was repeated three times. The resulting precipitate wasdissolved in methylene chloride and the solution was evaporated andpumped to giveD-Lac(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)meso-DAP-(D)-GlyOH (3) (932mg) as an amorphous solid.

N.M.R. (CDCl₃ -CD₃ oD), δ(ppm): 1.38 (3H, d, J=7 Hz), 1.47 (9H, s), 1.49(3H, d, J=7 Hz), 1.2-2.0 (6H, m), 2.0-2.5 (4H, m), 2.15 (3H, s), 3.99(2H, broad s), 5.02 (1H, q, J=7 Hz), 5.20 (2H, s), 7.39 (5H, s).

EXAMPLE 141 ##STR540##

1N Aqueous sodium hydroxide (4.6 ml) was added to a solution ofD-Lac-(oAc)-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-meso-DAP-(D)-GlyOH (1)(865 mg) in a mixture of methanol (8 ml) and water (4 ml) was stirred atambient temperature for two hours.

The reaction mixture was cooled to 0° C., neutralized to pH 7 with 1Naqueous hydrochloric acid and then concentrated. The concentrate wasdiluted with water and washed with ether. The aqueous layer wasconcentrated and the concentrate was acidified to pH 2 with 1N aqueoushydrochloric acid and then chromatographed on a macroporous non-ionicadsorption resin, HP20 (40 ml) eluting with 50% aqueous methanol.

The eluate was concentrated, triturated in ether and then evaporated togive D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-GlyOH (2) (600mg).

N.M.R. (D₂ O), δ(ppm): 1.0-2.8 (16H, m), 1.43 (9H, s), 3.83-4.5 (4H, m),3.96 (2H, s).

EXAMPLE 142 ##STR541##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-meso-DAP-(D)-GlyOH (1) (560 mg)was dissolved in tetrahydrofuran (5 ml) and the solution was stired atambient temperature for 15 minutes. The reaction mixture was evaporatedto dryness. The residue was triturated with ether and the supernatantwas discarded. The residue thus obtained was pumped to give a solidmaterials (647 mg). The solid materials were dissolved in water and thesolution was adjusted to pH 3.0 with 1N aqueous hydrochloric acid andchromatographed on a macroporous non-ionic adsorption resin, HP20 (40ml). The fractions containing the object compound were collected,concentrated and then lyophilized to giveD-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-meso-DAP(D)GlyOH (2) (324 mg).

N.M.R. (D₂ O), δ(ppm): 1.1-2.5 (10H, m), 1.37 (3H, d, J=7 Hz), 1.43 (3H,d, J=7 Hz), 4.01 (2H, s), 4.09 (1H, t, J=6 Hz), 4.2-4.6 (4H, m).

[α]_(D) ²⁵ =-35.4° (C=0.26, water).

EXAMPLE 143 ##STR542##

To a solution of n-octanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-D-AlaOH (1) (150mg) in methanol (150 ml) was added conc. hydrochloric acid (1 ml) wasadded at ambient temperature. The mixture was stood for two weeks at thesame temperature. To the reaction mixture was added triethylamine inorder to adjust the pH of the solution to 7 to 8. Evaporation of thesolvent in vacuo gave an oily residue, which was extracted with ethylacetate. The extract was washed with water and brine, and then driedover magnesium sulfate. After evaporation of the solvent in vacuo, theresidue was pulverized with isopropyl ether to given-octanoyl-γ-D-Glu(α-oMe)-(L)-mesoDAP-D-AlaoMe-(D)-oMe (2) (102 mg).

NMR (CDCl₃), δ(ppm): 0.90 (3H, t, J=7 Hz), 1.00-2.33 (25H, m), 3.73 (9H,s), 4.33-4.66 (4H, m), 6.50-7.50 (5H, m).

EXAMPLE 144

(1) Step 1

DL-2,3-Diacetoxypropionyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Z-(D)-mesoDAP-(D)-NHNHZ-(L)-GlyOHwas prepared substantially in the same manner as that of Step 1 ofExample 1.

NMR (CDCl₃), δ(ppm): 1.36 (3H, d, J=7 Hz), 2.01 (3H, s), 2.12 (3H, s),3.95 (2H, s), 4.25-4.65 (6H, m), 5.03-5.30 (8H, m), 7.35 (15H, s).

(2) Step 2

DL-2,3-Diacetoxypropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH wasprepared substantially in the same manner as that of Step 3 of Example27.

NMR (D₂ O), δ(ppm): 1.42 (3H, d, J=7 Hz), 2.10 (3H, s), 2.21 (3H, s),3.83 (1H, m), 3.96 (2H, s), 5.30 (1H, t, J=5 Hz).

(3) Step 3

DL-2,3-Dihydroxypropionyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH wasprepared substantially in the same manner as that of Step 2 of Example12.

NMR (D₂ O), δ(ppm): 1.47 (3H, d, J=7 Hz), 3.85 (2H, d, J=4 Hz), 4.00(2H, s), 4.27-4.63 (5H, m).

EXAMPLE 145

(1) Step 1

Stearoyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH wasprepared substantially in the same manner as that of Step 1 of Example27.

NMR (DMSO-d₆), δ(ppm): 0.80-2.40 (60H, m), 4.10-4.60 (5H, m), 5.12 (2H,s), 7.33 (B 5H, s), 7.80-8.43 (4H, m), 6.80-7.10 (1H, m).

(2) Step 2

Stearoyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaoME-(D)-oMewas prepared substantially in the same manner as that of Step 1 ofExample 131.

NMR (CDCl₃). δ(ppm): 0.68-2.53 (65H, m), 3.67 (3H, s), 3.70 (3H, s),4.10-4.70 (5H, m), 5.13 (2H, s), 6.33-6.67 (1H, m), 7.00 (1H, d, J=8Hz), 7.30 (5H, s), 7.30-7.70 (2H, m).

(3) Step 3

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaoMe-(D)-oMe wasprepared substantially in the same manner as that of Step 2 of Example131.

NMR (DMSO-d₆), δ(ppm): 0.68-2.50 (51H, m), 3.68 (3H, s), 3.77 (3H, s),4.0-4.70 (5H, m), 7.80-8.50 (5H, m).

EXAMPLE 146

(1) Step 1

Stearoyl-L-Ala-γ-D-Glu(α-oMe)-(L)-Boc-(D)-mesoDAP-(D)-oMe-(L)-D-AlaoMewas prepared substantially in the same manner as that of Step 1 ofExample 131.

NMR (CDCl₃), δ(ppm): 0.80-2.68 (60H, m), 3.90 (9H, s), 4.33-4.66 (5H,m).

(2) Step 2

Stearoyl-L-Ala-γ-D-Glu(α-oMe)-(L)-mesoDAP-(D)-oMe-(L)-D-AlaoMe wasprepared substantially in the same manner as that of Step 2 of Example131.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.68-2.50 (51H, m), 3.87 (6H, s), 3.90 (3H,s), 4.27-4.66 (5H, m).

EXAMPLE 147

(1) Step 1

Stearoyl-L-Val-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH wasprepared substantially in the same manner as that of Step 1 of Example27.

NMR (DMSO-d₆), δ(ppm): 0.70-2.50 (64H, m), 4.0-4.60 (5H, m), 5.15 (2H,s), 6.70-7.10 (1H, m), 7.36 (5H, s), 7.50-8.50 (4H, m).

(2) Step 2

Stearoyl-L-Val-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH was preparedsubstantially in the same manner as that of Step 3 of Example 27.

NMR (DMSO-d₆), δ(ppm): 0.68-2.50 (55H, m), 3.67-3.90 (1H, m), 4.0-4.50(4H, m), 7.50-8.40 (4H, m).

PREPARATION 115

(1) Step 1 ##STR543##

Stearoyl-L-Ser(oBzl)-D-Glu(α-OBzl) (2) was prepared substantially in thesame manner as Preparation 85.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.90 (3H, m), 1.1-1.9 (30H, m), 1.95-2.65(6H, m), 3.60-3.90 (3H, m), 4.32 (2H, s), 5.19 (2H, s), 7.35 (10H, s).

(2) Step 2 ##STR544##

Stearoyl-L-Ser(oBzl)-D-Glu(α-oBzl)-γ-OSu (3) was prepared substantiallyin the same manner as Preparation 86.

NMR (CDCl₃, δ): 0.89 (3H, m), 1.28 (30H, s), 2.81 (4H, s), 4.56 (2H, s),5.20 (2H, s), 7.33 (5H, s), 7.36 (5H, s).

Preparation 116

(1) Step 1 ##STR545##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaoBzl (1) (11.0 g) was dissolved inmethanol (100 ml) and the solution of diazomethane in ether was addedthereto. After stirring for 15 minutes, the resultant mixture wastreated with acetic acid, concentrated in vacuo. The residue wasdissolved in ethyl acetate and washed with 3% sodium bicarbonate, driedover magnesium sulfate and then concentrated in vacuo to giveZ-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(oBzl)-(D)-OMe (2) (9.1 g).

NMR (CDCl₃), δ(ppm): 1.3-2.0 (18H, m), 3.72 (3H, s), 4.32 (1H, m), 4.82(1H, t, J=7), 5.12 (2H, s), 5.18 (2H, s), 5.59 (1H, d, J=8), 6.82 (1H,d, J=8), 7.37 (10H, s).

(2) Step 2 ##STR546##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(oBzl)-(D)-OMe (2) (9.1 g) was dissolvedin a mixture of methanol (100 ml) and water (20 ml), and hydrogenatedunder 32 atmospheric pressure of hydrogen over 10% palladium-carbon.After removal of the catalyst, the mixture was concentrated and treatedwith ether to give a crystalline mass (5.7 g), which was purified byusing HP20 to give Boc-(D)-mesoDAP-(L)-D-AlaOH-(D)-OMe (3) (5.02 g).

NMR (CDCl₃), δ(ppm): 1.30-2.00 (18H, m), 3.72 (3H, s), 3.90-4.45 (3H,m).

Preparation 117 ##STR547##

Z-(D)-mesoDAP-(D)-NHNHZ-(L)-D-AlaOH (2) was prepared substantially inthe same manner as Preparation 63.

mp. 161°-165° C. (dec.)

Preparation 118

(1) Step 1 ##STR548##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe-(D)-NHNHBoc (2) was preparedsubstantially in the same manner as step 1 of Preparation 53.

NMR (DMSO-d₆), δ(ppm): 1.05-1.95 (2H, m), 3.65 (3H, s), 3.70-4.65 (3H,m), 5.08 (2H, s), 6.70 (1H, m), 7.35 (5H, s), 8.34 (2H, m), 8.68 (1H,m), 9.61 (1H, s).

(2) Step 2 ##STR549##

Z-(L)-mesoDAP-(L)-D-AlaOMe (3) was prepared substantially in the samemanner as step 2 of Preparation 53.

NMR (CD₃ OD), δ(ppm): 1.35 (3H, d, J=7 Hz), 1.45-2.10 (6H, m), 3.71 (3H,s), 4.15 (1H, m), 4.42 (1H, q, J=7 Hz), 5.11 (2H, s), 7.36 (5H, s).

(3) Step 3 ##STR550##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe (4) was prepared substantially in thesame manner as step 3 of Preparation 53.

NMR (CD₃ OD), δ(ppm): 1.35 (3H, d, J=7 Hz), 1.43 (9H, s), 3.69 (3H, s),4.16 (1H, m), 4.41 (1H, q, J=7 Hz), 5.10 (2H, s), 7.34 (5H, s).

(4) Step 4 ##STR551##

Boc-(D)-mesoDAP-(L)-D-AlaOMe (5) was prepared substantially in the samemanner as step 4 of Preparation 53.

NMR (CD₃ OD), δ(ppm): 1.2-2.0 (18H, m), 3.77 (3H, s), 3.7-4.1 (2H, m),4.51 (1H, q, J=7 Hz).

Preparation 119

(1) Step 1 ##STR552##

Stearoyl-L-Ala-D-GluOMe (2) was prepared substantially in the samemanner as Preparation 85.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.90 (3H, m), 1.30 (30H, s), 1.42 (9H, s),3.79 (3H, s).

(2) Step 2 ##STR553##

Stearoyl-L-Ala-D-Glu(α-OMe)(γ-OSu) (3) was prepared substantially in thesame manner as Preparation 86.

NMR (CDCl₃), δ(ppm): 0.89 (3H, m), 1.30 (30H, s), 1.45 (9H, s), 2.88(4H, s), 4.4-4.9 (2H, m), 6.61 (1H, d, J=7 Hz), 7.57 (1H, d, J=7 Hz).

Preparation 120 ##STR554##

Boc-L-Ala-D-Glu(α-OBzl)(1)(1.68 g) was added to trifluoroacetic acid (10ml) and the mixture was stirred for 15 minutes at ambient temperature.After evaporation of trifluoroacetic acid, the residue was dissolved indioxane (30 ml). The solution was neutralized with triethylamine andthen a solution of D-LacOSu (0.77 g) in methyl cyanide (10 ml) was addedthereto. The resulting mixture was stirred for 5 hours at ambienttemperature and the solvent was removed under reduced pressure. Theresidue was dissolved in ethyl acetate and the ethyl acetate layer waswashed with water (30 ml×2), dried over magnesium sulfate and thenconcentrated under reduced pressure to give an oil. The oil waschromatographed on silica gel and eluted with a mixture of chloroformand methanol (20:1) to give D-Lac-L-Ala-D-Glu(α-OBzl) (2)(0.77 g).

NMR (CDCl₃), δ(ppm): 1.40 (6H, d, J=7 Hz), 1.8-2.6 (4H, m), 4.2-4.8 (3H,m), 5.17 (2H, s), 7.35 (5H, s).

Preparation 121

(1) Step 1 ##STR555##

Phenylacetyl-D-Glu(α-OBzl)(2) was prepared substantially in the samemanner as Preparation 85.

NMR (CDCl₃), δ(ppm): 1.80-2.50 (4H, m), 3.50 (2H, s), 4.50-4.90 (1H, m),5.13 (2H, s), 6.33 (1H, d, J=8 Hz), 7.26 (5H, s), 7.33 (5H, s), 9.76(1H, s).

(2) Step 2 ##STR556##

Phenylacetyl-D-Glu(α-OBzl)-γ-OSu (3) was prepared substantially in thesame manner as Preparation 86.

NMR (CDCl₃), δ(ppm): 2.06-2.72 (4H, m), 2.76 (4H, s), 3.66 (2H, s),4.60-4.88 (1H, m), 5.20 (2H, s), 6.64 (1H, d, J=8 Hz), 7.40 (5H, s),7.48 (5H, s).

Preparation 122

(1) Step 1 ##STR557##

Phenylacetyl-L-Ala-D-Glu(α-OBzl) (2) was prepared substantially in thesame manner as Preparation 85.

NMR (CDCl₃), δ(ppm): 1.23 (3H, d, J=7 Hz), 1.80-2.50 (4H, m), 3.53 (2H,s), 4.33-4.80 (2H, m), 5.10 (2H, s), 6.90 (1H, d, J=7 Hz), 7.30 (5H, s),7.40 (5H, s), 7.60 (1H, d, J=7 Hz), 8.83 (1H, s).

(2) Step 2 ##STR558##

Phenylacetyl-L-Ala-D-Glu(α-OBzl)-γ-OSu (3) was prepared substantially inthe same manner as Preparation 86.

NMR (CDCl₃), δ(ppm): 1.23 (3H, d, J=7 Hz), 1.84-2.67 (4H, m), 2.76 (4H,s), 3.53 (2H, s), 4.40-4.86 (2H, m), 5.13 (2H, s), 6.40 (1H, d, J=8 Hz),7.27 (5H, s), 7.33 (5H, s).

Preparation 123

(1) Step 1 ##STR559##

n-Octanoyl-L-Ala-D-Glu(α-OBzl)(2) was prepared substantially in the samemanner as Preparation 85.

NMR (CDCl₃), δ(ppm): 0.68-2.50 (22H, m), 4.40-4.80 (2H, m), 5.15 (2H,s), 6.70 (1H, d, J=7 Hz), 7.30 (5H, s), 7.54 (1H, d, J=7 Hz), 9.50 (1H,s).

(2) Step 2 ##STR560##

n-Octanoyl-L-Ala-D-Glu(α-OBzl)-γ-OSu (3) was prepared substantially inthe same manner as Preparation 86.

NMR (CDCl₃), δ(ppm): 0.92 (3H, t, J=7 Hz), 1.10-1.80 (15H, m), 2.0-2.80(4H, m), 2.82 (4H, s), 4.50-4.84 (2H, m), 5.24 (2H, s), 6.68 (1H, d, J=8Hz), 7.44 (5H, s), 7.68 (1H, d, J=8 Hz).

Preparation 124

(1) Step 1 ##STR561##

n-Octanoyl-D-Glu(α-OBzl) (2) was prepared substantially in the samemanner as Preparation 85.

NMR (CDCl₃), δ(ppm): 0.87 (3H, t, J=7 Hz), 1.0-2.60 (16H, m), 4.50-5.00(1H, m), 5.20 (2H, s), 6.35 (1H, d, J=7 Hz), 7.40 (5H, s), and 9.90 (1H,s).

(2) Step 2 ##STR562##

n-Octanoyl-D-Glu(α-OBzl)-γ-OSu (3) was prepared substantially in thesame manner as Preparation 86.

NMR (CDCl₃), δ(ppm): 0.90 (3H, t, J=7 Hz), 1.20-1.80 (12H, m), 2.16-2.80(4H, m), 2.86 (4H, s), 4.68-4.96 (1H, m), 5.62 (2H, s), 6.52 (1H, d, J=8Hz), 7.48 (5H, s).

Preparation 125 ##STR563##

Boc-(D)-mesoDAP-(D)-NHNHBoc (2) was prepared substantially in the samemanner in Preparation 20.

NMR (CD₃ OD-D₂ O), δ(ppm): 1.50-2.20 (24H, m), 3.60-3.90 (1H, m),4.00-4.20 (1H, m).

EXAMPLE 148

(1) Step 1 ##STR564##

Lauroyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(OH) (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (DMSO-d₆): δ, 0.80-2.50 (48H, m), 4.0-4.50 (5H, m), 5.10 (2H, s),6.80-7.00 (1H, m), 7.33 (5H, s), 7.70-8.40 (4H, m).

(2) Step 2 ##STR565##

Lauroyl-L-Ala-γ-D-Glu(α-oMe)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(oMe)-(D)-oMe(4) was prepared substantially in the same manner as step 1 of Example131.

NMR (CDCl₃): δ, 0.80-2.50 (48H, m), 3.73 (9H, s), 4.10-4.70 (5H, m),5.30 (1H, d, J=8 Hz), 6.50 (1H, broad s), 6.96 (1H, d, J=8 Hz),7.33-7.65 (2H, m).

(3) Step 3 ##STR566##

Lauroyl-L-Ala-γ-D-Glu(α-oMe)-(L)-mesoDAP-(L)-D-Ala(oMe)-(D)-oMehydrochloric acid salt (5) was prepared substantially in the same manneras step 1 of Example 131.

NMR (DMSO-d₆): δ, 0.80-2.40 (39H, m), 3.68 (6H, s), 3.78 (3H, s),3.88-4.00 (1H, m), 4.12-4.50 (4H, m), 8.04 (2H, t, J=8 Hz), 8.20 (2H, t,J=8 Hz), 8.64 (2H, broad).

EXAMPLE 149

(1) Step 1 ##STR567##

Stearoyl-L-Ser(oBzl)-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (3)was prepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.88 (3H, m), 1.1-1.4 (30H, m), 1.45 (9H,s), 3.6-3.9 (2H, m), 4.53 (2H, s), 5.15 (2H, s), 7.31 (10H, s). (2) Step2 ##STR568##

Stearoyl-L-Ser-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(OH) (4) wasprepared substantially in the same manner as step 2 of Example 96.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.90 (3H, m), 1.30 (30H, s), 1.46 (9H, s),2.1-2.55 (4H, m), 3.65-4.80 (7H, m).

(3) Step 3 ##STR569##

Stearoyl-L-Ser-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-Ala(OH) (5) was preparedsubstantially in the same manner as step 3 of Example 96.

NMR (CDCl₃, CD₃ OD), δ(ppm): 0.90 (3H, m), 1.05-2.60 (45H, m), 4.02 (1H,m), 4.30-4.95 (6H, m).

EXAMPLE 150

(1) Step 1 ##STR570##

Stearoyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-(D)-oMe (3)was prepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.90 (3H, s), 1.28 (30H, s), 1.40 (9H, s),3.74 (3H, s), 4.05-4.60 (5H, m), 5.18 (2H, s), 7.36 (5H, s).

(2) Step 2 ##STR571##

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(OH)-(D)-oMe(4) was prepared substantially in the same manner as step 2 of Example96.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.89 (3H, m), 1.30 (30H, s), 1.46 (9H, s),3.77 (3H, s), 4.05-4.60 (5H, m).

(3) Step 3 ##STR572##

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-Ala(OH)-(D)-oMehydrochloric acid salt (5) was prepared substantially in the same manneras step 3 of Example 96.

NMR (DMSO-d₆), δ(ppm): 0.85 (3H, m), 3.74 (3H, s), 3.8-4.55 (5H, m).

EXAMPLE 151

(1) Step 1 ##STR573##

Stearoyl-L-Ala-γ-D-Glu(α-oBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.88 (3H, m), 1.27 (30H, s), 1.42 (9H, s),3.71 (3H, s), 4.03-4.65 (5H, m), 5.18 (2H, s), 7.35 (5H, s).

(2) Step 2 ##STR574##

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe (4) wasprepared substantially in the same manner as step 2 of Example 96.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.88 (3H, m), 1.31 (30H, s), 1.46 (9H, s),3.73 (3H, s), 4.00-4.65 (5H, m).

(3) Step 3 ##STR575##

Stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOMe hydrochloric acidsalt (5) was prepared substantially in the same manner as step 2 ofExample 131.

NMR (DMSO-d₆), δ(ppm): 0.86 (3H, m), 3.68 (3H, s), 3.85 (1H, s),4.10-4.50 (4H, m).

EXAMPLE 152

(1) Step 1 ##STR576##

Stearoyl-L-Ala-γ-D-Glu(α-OMe)-(L)-Boc(D)-mesoDAP-(L)-D-AlaOH (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.89 (3H, m), 1.28 (30H, s), 1.45 (9H, s),3.76 (3H, s).

(2) Step 2 ##STR577##

Stearoyl-L-Ala-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-D-AlaOH hydrochloric acidsalt (4) was prepared substantially in the same manner as step 2 ofExample 131.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.98 (3H, m), 1.0-2.5 (38H, m), 3.73 (3H,s), 3.94 (1H, m), 4.20-4.57 (4H, m).

EXAMPLE 153

(1) Step 1 ##STR578##

Stearoyl-L-Ala-γ-D-Glu(α-OMe)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-(D)-OMe (3)was prepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.92 (3H, m), 1.31 (30H, s), 1.48 (9H, s),3.77 (6H, s).

(2) Step 2 ##STR579##

Stearoyl-L-Ala-γ-D-Glu (α-oMe)-(L)-mesoDAP-(L)-D-Ala-(D)-OMehydrochloric acid salt (4) was prepared substantially in the same manneras step 2 of Example 131.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.90 (3H, m), 1.08-2.47 (48H, m), 3.74 (3H,s), 3.85 (3H, s), 3.93-4.09 (1H, m), 4.25-4.80 (4H, m).

EXAMPLE 154

(1) Step 1 ##STR580##

Stearoyl-L-Ala-γ-D-Glu(α-oMe)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.88 (3H, m), 1.28 (30H, s), 1.46 (9H, s),3.75 (6H, s).

(2) Step 2 ##STR581##

Stearoyl-L-Ala-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-D-AlaOMe hydrochloric acidsalt (4) was prepared substantially in the same manner as step 2 ofExample 131.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.89 (3H, m), 1.0-2.5 (48H, m), 3.75 (6H,s), 3.95 (1H, m), 4.15-4.95 (4H, m).

EXAMPLE 155

(1) Step 1 ##STR582##

Phenylacetyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3)was prepared substantially in the same manner as step 1 of Example 125.

NMR (CD₃ OD), δ(ppm): 1.47 (18H, s), 3.60 (2H, s), 5.20 (2H, s), 7.33(5H, s), 7.40 (5H, s).

(2) Step 2 ##STR583##

Phenylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4) wasprepared substantially in the same manner as step 2 of Example 96.

NMR (CD₃ OD), δ(ppm): 1.47 (18H, s), 3.60 (2H, s), 4.00-4.66 (4H, s),7.30 (5H, s).

(3) Step 3 ##STR584##

Phenylacetyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP (5) was preparedsubstantially in the same manner as step 3 of Example 96.

NMR (D₂ O), δ(ppm): 1.40 (3H, d, J=8 Hz), 3.60 (2H, s), 3.80 (1H, m),4.24 (2H, t, J=7 Hz), 7.36 (5H, s).

EXAMPLE 156

(1) Step 1 ##STR585##

Phenylacetyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CD₃ OD), δ(ppm): 1.47 (18H, s), 3.65 (2H, broad s), 5.17 (2H, s),7.30 (5H, s), 7.36 (5H, s).

(2) Step 2 ##STR586##

Phenylacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (4) wasprepared substantially in the same manner as step 2 of Example 96.

(3) Step 3 ##STR587##

Phenylacetyl-γ-D-Glu(α-OH)-(L)-mesoDAP (5) was prepared substantially inthe same manner as step 3 of Example 96.

NMR (D₂ O), δ(ppm): 1.20-2.50 (10H, m), 3.64 (2H, s), 3.72 (1H, t, J=7Hz), 4.24 (1H, t, J=7 Hz), 4.30 (1H, t, J=7 Hz), 5.36 (5H, s).

EXAMPLE 157

(1) Step 1 ##STR588##

(12-Hydroxy)stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH(2) was prepared substantially in the same manner as step 1 of Example1.

NMR (CD₃ OD), δ(ppm): 0.90 (3H, t, J=5 Hz), 1.25 (9H, s), 1.15-1.83(39H, m) 2.15-2.42 (4H, m), 3.93 (2H, s), 4.25-4.53 (5H, m).

(2) Step 2 ##STR589##

(12-Hydroxy)stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (3) wasprepared substantially in the same manner as step 2 of Example 1.

NMR (DMSO-d₆), δ(ppm): 0.83 (3H, t, J=5 Hz), 1.16-1.66 (39H, m),1.92-2.25 (4H, m), 3.73 (2H, broad s), 4.07-4.25 (5H, m).

EXAMPLE 158

(1) Step 1 ##STR590##

α-D,L-Hydroxypalmitoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH(2) was prepared substantially in the same manner as step 1 of Example1.

NMR (CD₃ OD), δ(ppm): 0.92 (3H, t, J=5 Hz), 1.30-1.75 (37H, m), 1.45(9H, s), 2.30-2.63 (2H, m), 3.97 (2H, s), 4.00-4.58 (5H, m).

(2) Step 2 ##STR591##

α-D,L-Hydroxypalmitoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (3) wasprepared substantially in the same manner as step 2 of Example 1.

NMR (DMSO-d₆), δ(ppm): 8.66 (3H, t, J=5 Hz), 1.23-1.75 (37H, m),2.08-2.23 (2H, m), 3.63 (2H, broad s), 4.13-4.43 (5H, m).

EXAMPLE 159

(1) Step 1 ##STR592##

(12-D,L-Acetoxy)stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc-(L)-GlyOH(2) was prepared substantially in the same manner as step 1 of Example1.

NMR (CD₃ OD), δ(ppm): 0.90 (3H, t, J=5 Hz), 1.30-1.73 (37H, m), 1.43(9H, s), 2.02 (3H, s), 2.13-2.36 (4H, m), 3.95 (2H, s), 4.33-4.53 (5H,m).

(2) Step 2 ##STR593##

(12-D,L-Acetoxy)stearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (3)was prepared substantially in the same manner as step 2 of Example 1.

NMR (CD₃ OD), δ(ppm): 0.87 (3H, t, J=5 Hz), 1.28-1.73 (37H, m), 2.00(3H, s), 2.13-2.33 (4H, m), 3.92 (2H, s), 4.30-4.50 (5H, m).

EXAMPLE 160

(1) Step 1 ##STR594##

D-Lac-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CD₃ OD), δ(ppm): 1.37 (6H, d, J=7 Hz), 1.3-2.5 (10H, m), 3.67 (3H,s), 3.70 (3H, s), 3.95 (2H, s), 4.0-4.6 (5H, m), 5.09 (2H, s), 5.17 (2H,s), 7.35 (10H, s).

(2) Step 2 ##STR595##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOMe-(D)-OMe (4) wasprepared substantially in the same manner as Example 73.

NMR (D₂ O), δ(ppm): 1.37 (3H, d, J=6 Hz), 1.41 (3H, d, J=6 Hz), 1.5-2.4(10H, m), 3.73 (3H, s), 3.82 (3H, s), 4.00 (2H, s), 4.0-4.5 (5H, m).

EXAMPLE 161

(1) Step 1 ##STR596##

Phenylacetyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-(D)-NHNHBoc(3) was prepared substantially in the same manner as step 1 of Example125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 1.48 (18H, s), 3.62 (2H, s), 3.67 (1H, m),5.19 (2H, s), 7.34 (5H, s), 7.39 (5H, s).

(2) Step 2 ##STR597##

Phenylacetyl-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-(D)-NHNHBoc (4)was prepared substantially in the same manner as step 2 of Example 89.

NMR (DMSO-d₆), δ(ppm): 1.30 (3H, d, J=7), 3.55 (2H, s), 3.8-4.8 (4H, m),6.77 (1H, d, J=8), 7.36 (5H, s), 7.8-8.9 (5H, m), 8.63 (1H, s).

(3) Step 3 ##STR598##

Phenylacetyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was preparedsubstantially in the same manner as step 3 of Example 89.

NMR (D₂ O), δ(ppm): 1.39 (3H, d, J=7 Hz), 1.20-2.55 (10H, m), 3.66 (2H,s), 3.80 (1H, t, J=6 Hz), 4.20-4.55 (3H, m), 7.36 (5H, s).

EXAMPLE 162

(1) Step 1 ##STR599##

n-Octanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-(D)-NHNHBoc(3) was prepared substantially in the same manner as step 1 of Example125.

NMR (CDCl₃ -CD₃ OD), δ(ppm): 0.90 (3H, m), 1.15-2.53 (46H, m), 4.27-4.69(5H, m), 5.21 (2H, s), 7.40 (5H, s).

(2) Step 2 ##STR600##

n-Octanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-(D)-NHNHBoc(4) was prepared substantially in the same manner as step 2 of Example96.

NMR (DMSO-d₆), δ(ppm): 0.6-2.4 (49H, m), 3.8-4.6 (5H, m), 6.71 (1H, m),7.75-8.80 (5H, m), 9.60 (1H, s).

(3) Step 3 ##STR601##

n-Octanoyl-D-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was preparedsubstantially in the same manner as step 3 of Example 96.

NMR (D₂ O), δ(ppm): 0.87 (3H, m), 1.1-2.6 (28H, m), 3.85 (1H, t, J=6),4.2-4.6 (4H, m).

EXAMPLE 163

(1) Step 1 ##STR602##

n-Octanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃), δ(ppm): 0.68-2.50 (46H, m), 4.16-4.83 (4H, m), 5.14 (2H,s), 6.70-7.0 (broad, 1H), 7.36 (5H, s), 7.70-8.0 (broad, 1H), 9.20 (1H,s).

(2) Step 2 ##STR603##

n-Octanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP (4) was preparedsubstantially in the same manner as steps 2 and 3 of Example 96.

NMR (D₂ O), δ(ppm): 0.68-2.50 (28H, m), 3.67-4.00 (1H, m), 4.16-4.50(3H, m).

EXAMPLE 164

(1) Step 1 ##STR604##n-Octanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(D)-NHNHBoc (3) wasprepared substantially in the same manner as step 1 of Example 125.

NMR (CDCl₃), δ(ppm): 0.70-2.50 (43H, m), 4.0-4.80 (3H, m), 5.20 (2H, s),5.50-5.80 (1H, m), 6.30-6.60 (2H, m), 6.80-7.10 (2H, m), 7.35 (5H, s),9.10 (1H, s).

(2) Step 2 ##STR605##

n-Octanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP (4) was prepared substantially inthe same manner as steps 2 and 3 of Example 96.

NMR (D₂ O), δ(ppm): 0.68-2.60 (25H, m), 3.80 (1H, t, J=7 Hz), 4.16-4.50(2H, m).

Preparation 126 ##STR606##

D-AlaOH (2)(1.78 g) was dissolved in a mixture of water (40 ml), dioxane(40 ml) and triethylamine (4.04 g). To this solution was addedBoc-L-Lys(ε-Z)OSu (1)(9.26 g) and the resulting solution was leftovernight at ambient temperature and then filtered.

The filtrate was evaporated to give an oily residue which was dissolvedin water. The solution was acidified with dil hydrochloric acid and thenextracted with ethyl acetate. The organic layer was washed with water,dried over magnesium sulfate and then evaporated to give a white foam.The foam was dissolved in trifluoroacetic acid (30 ml) and reacted for30 minutes at ambient temperature. Excess trifluoroacetic acid wasevaporated to give a paste which was dissolved in water. The solutionwas passed through HP20 column.

The column was eluted with water and water-methanol (1:1), successively.The latter fractions were combined and evaporated to giveL-Lys(ε-Z)-D-AlaOH (3) (4.50 g).

IR (Nujol): 3350, 3300, 1685, 1660, 1640 cm⁻¹.

NMR (CD₃ OD): δ 1.30 (3H, d, J=7 Hz), 1.20-1.70 (6H, m), 2.86-3.50 (2H,m), 4.16 (1H, q, J=7 Hz), 5.00 (2H, s), 7.30 (5H, s).

Preparation 127 ##STR607##

Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe (1)(0.66 g) was dissolved inmethanol (10 ml) and hydrogenated over 10% palladium charcoal (0.12 g).After removal of the catalyst by filtration, the filtrate wasconcentrated under reduced pressure to giveBoc-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe (2)(0.48 g).

IR (CH₂ Cl₂): 3400, 3360, 1735, 1705, 1670 cm⁻¹.

NMR (CDCl₃) δ: 1.42 (9H, s), 1.3-2.2 (6H, m), 3.3-3.6 (1H, m), 3.77 (6H,s), 4.05 (2H, d, J=5 Hz), 4.1-4.5 (1H, m).

Preparation 128 ##STR608##

Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe-(D)-NHNHBoc (1)(1.8 g) was added totrifluoroacetic acid and the mixture was stirred for 15 minutes at roomtemperature. After evaporation of the solvent, the residue was dissolvedin methanol (50 ml) and treated with N-bromosuccinimide (1.58 g), andthe mixture was stirred for 10 minutes at 0°-5° C. and concentrated invacuo. The residue was dissolved in water (40 ml) and the excess reagentwas decomposed by adding 5% sodium bisulfite. The mixture wasneutralized to pH 9 with sodium bicarbonate, and extracted with ethylacetate (40 ml). The extract was washed with water and dried overmagnesium sulfate, and the solvent was removed in vacuo. The residue wasdissolved in a mixture of dioxane (20 ml) and water (10 ml). To thismixture triethylamine (0.66 g) and di-t-butyldicarbonate (1.42 g) wasadded and stirred for an hour at room temperature. After evaporation ofthe solvent, the residue was dissolved in ethylacetate (40 ml), andwashed successively with 2.5% hydrochloric acid (15 ml) and water (20ml), dried over magnesium sulfate and evaporated. The residue waspulverized with isopropylether to giveZ-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe (2)(0.7 g).

NMR (CD₃ OD) δ: 1.43 (9H, s), 1.4-2.0 (6H, m), 3.72 (6H, s), 3.8-4.3(4H, m), 5.12 (2H, s), 7.35 (5H, s).

Preparation 129 ##STR609##

To a mixture of Z-(D)-mesoDAP-(D)-OMe (1)(90 mg) and triethylamine (50mg) in 50% aqueous dioxane was added di-t-butyldicarbonate (120 mg) andthe mixture was stirred for overnight at room temperature. Afterevaporation of dioxane, the aqueous solution was washed with ether (10ml) and acidified to pH3 with 5% hydrochloric acid and extracted wihethyl acetate (30 ml). The extract was washed with water and dried overmagnesium sulfate. The solvent was removed under reduced pressure togive Boc-(L)-Z-(D)-mesoDAP-(D)-OMe (2)(110 mg).

IRcm⁻¹ in CH₂ Cl₂ : 3390, 1730 (shoulder), 1710.

NMR (CDCl₃) δ: 1.45 (9H, s), 1.4-2.0 (6H, m), 3.68 (3H, s), 4.0-4.4 (2H,m), 5.08 (2H, s), 7.30 (5H, s).

Preparation 130 ##STR610##

To a solution of diZDAP(D)-OMe (1)(2.20 g) in benzene (50 ml) was addedthionyl chloride (5 ml) and the mixture was refluxed for an hour. Thereaction mixture was concentrated under reduced pressure. The residualoil was dissolved in a mixture of dioxane (20 ml) and water (20 ml) andthen stood overnight at room temperature. After concentration ofdioxane, the concentrate was washed with ethyl acetate (20 ml). Theaqueous layer was concentrated to about 10 ml and neutralized to pH 5with 5% aqueous sodium bicarbonate. The resulting crystalline solid wasfiltered and washed with water to give Z(D)-mesoDAP-(D)-OMe (2)(0.58 g).

IR cm⁻¹ (Nujol): 3100, 1745, 1695, 1610.

NMR (DCl-D₂ O), δ: 1.3-2.3 (6H, m), 3.73 (3H, s), 4.0-4.5 (2H, m), 5.15(2H, s), 7.43 (5H, s).

Preparation 131 ##STR611##

Z-(D)-mesoDAP-(D)-OEt (2) was prepared substantially in the same manneras Preparation 130.

IR cm⁻¹ (Nujol): 3300-2200, 1725, 1690, 1615.

NMR (D₂ O-DCl), δ: 1.25 (3H, t, J=7 Hz), 1.5-2.2 (6H, m), 3.9-4.5 (4H,m), 5.13 (2H, s), 7.42 (5H, s).

Preparation 132 ##STR612##

Into a solution of diZ-mesoDAP-(D)-NHNHBoc (1)(30.3 g) in ethyl acetate(240 ml) was passed a stream of hydrogen chloride gas for 40 minutesunder ice-bath cooling. The reaction mixture was stirred for an hour atthe same temperature. The precipitated crystals were filtered and washedwith ether to give diZ-mesoDAP-(D)-NHNH₂ -HCl (2)(24.2 g).

IR cm⁻¹ (Nujol): 3280, 3400-2200, 1720, 1680, 1660.

NMR (DMSO-d₆), δ: 1.3-2.0 (6H, m), 3.8-4.2 (2H, m), 5.04 (4H, s), 7.36(10H, s).

Preparation 133 ##STR613##

Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe-(D)-NHNHBoc (1)(9.57 g) was added totrifluoroacetic acid (96 ml) and the mixture was stirred for an hour atambient temperature. After evaporation of trifluoroacetic acid, theresidue was dissolved in 50% aqueous dioxane (180 ml) and the mixturewas treated with N-bromosuccinimide (6.43 g). After stirring for an hourunder ice-bath cooling, the excess reagent was decomposed with 10%sodium bisulfite. The resulting solution was adjusted to pH 8 with 50%sodium bicarbonate and then a solution of di-t-butylcarbonate (8.56 g)in dioxane (35 ml) was added thereto.

The resulting mixture was stirred for 20 hours at ambient temperatureand evaporated. The aqueous solution was washed with ethyl acetate (100ml) and adjusted to pH 3 with 5% hydrochloric acid and then extractedwith ethyl acetate (250 ml). The extract was washed with water and driedover magnesium sulfate. The solvent was removed under reduced pressureto leave an oil, which was chromatographed on silica gel (220 g) elutingwith a mixture of chloroform and methanol (20:1) to giveZ-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe (2)(4.78 g).

NMR (CD₂ OD), δ: 1.47 (9H, s), 1.4-2.0 (6H, m), 3.73 (3H, s), 3.97 (2H,s), 4.0-4.35 (2H, m), 5.13 (2H, s), 7.37 (5H, s).

Preparation 134 ##STR614##

A solution of Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe (1) (4.78 g) in a mixtureof methanol (100 ml) and water (15 ml) was hydrogenated over 10%palladium-charcoal (1.45 g). After removal of the catalyst, the filtratewas evaporated to dryness under reduced pressure. The residue waspulverized with ether to give Boc-(D)-mesoDAP-(L)-GlyOMe (2)(3.26 g).

IR cm⁻¹ (Nujol): 3600-2200, 1740, 1680, 1220, 1170, 1050, 1030, 860.

Preparation 135 ##STR615##

A solution of Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOBzl (1) (13.7 g) in methanol(140 ml) was treated with ethereal diazomethane under ice-bath cooling.The mixture was stirred for an hour at the same temperature andconcentrated under reduced pressure. The residue was pulverized withisopropylether to give Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOBzl-(D)-OMe(2)(11.9 g).

NMR (CD₃ OD), δ: 1.44 (9H, s), 1.3-2.0 (6H, m), 3.68 (3H, s), 3.96 (2H,d, J=4 Hz), 4.0-4.3 (2H, m), 5.06 (2H, s), 5.14 (2H, s), 7.30 (10H, s).

Preparation 136 ##STR616##

A solution of Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOBzl-(D)-OMe (1)(10.17 g) ina mixture of methanol (150 ml) and water (15 ml) was hydrogenated over10% palladium charcoal (3.0 g) for 4 hours. After removal of thecatalyst, the filtrate was evaporated to dryness under reduced pressure.The residue was pulverized with ether to giveBoc-(D)-mesoDAP-(L)-GlyOH-(D)-OMe (2)(5.85 g).

IR cm⁻¹ (Nujol): 3325, 1750, 1690, 1640, 1610.

NMR (CD₃ OD), δ: 1.40 (9H, s), 1.5-2.0 (6H, m), 3.70 (3H, s), 3.82 (2H,s), 3.8-4.2 (2H, m).

Preparation 137 ##STR617##

To a solution of n-docosonoyl-L-Ala-D-Glu(α-OBzl)(1) (4.76 g) in amixture of tetrahydrofuran (50 ml) and chloroform (100 ml) were addeddicyclohexylcarbodiimide (1.56 g) and N-hydroxysuccinimide (0.87 g). Themixture was kept for 17 hours at room temperature. The precipitate wasfiltered off and washed with chloroform (150 ml). The filtrate wasconcentrated in vacuo to give a crystal line residue, which wascollected and washed with diisopropylether to given-docosanoyl-L-Ala-D-Glu(α-OBzl)-OSu (2) (5.70 g).

IR (Nujol): 3380, 1820, 1790, 1750, 1640 cm⁻¹.

NMR (CDCl₃ -CD₃ OD), δ: 0.91 (3H, m), 1.06-2.53 (47H, m), 2.84 (4H, s),4.35-4.75 (2H, m), 5.19 (2H, s), 7.33 (5H, s).

Preparation 138 ##STR618##

Box-L-Ala-D-Glu(α-OBzl) (1) (4.0 g) was dissolved in trifluoroaceticacid (30 ml) and stirred for 2 hours at room temperature. The reactionmixture was concentrated in vacuo, and the residual oil was washed withethylether. The oil was dissolved in methylenechloride (40 ml) andtriethylamine (2.48 g) and methanol (12 ml) were added thereto. Afterthe solution turned clean, n-docosanoic acid succinimidoester (4.28 g)and another 0.5 equivalent mole of triethylamine were added. Afterstirring for 19 hours, the reaction mixture was concentrated in vacuo.Water (15 ml) and 1N-hydrochloric acid (30 ml) were added to thesolution to give a crystalline mass. The precipitate was collected andwashed with water (50 ml) to give a crude crystal (5.78 g), which waspurified by washing with hot diiso propylether to given-docosanoyl-L-Ala-D-Glu(α-OBzl) (2) (5.1 g).

IR (Nujol): 3300, 1725, 1700, 1650, 1630 cm⁻¹.

NMR (CDCl₃),: 0.88 (3H, m), 1.05-2.50 (47H, m), 4.3-4.8 (2H, m), 5.15(2H, s), 7.32 (5H, s).

Preparation 139 ##STR619##

n-Tetracosanoyl-L-Ala-D-Glu(α-OBzl)Osu (2) was prepared substantially inthe same manner as Preparation 137.

IR (Nujol): 3380, 1820, 1790, 1745, 1630 cm⁻¹.

NMR (CDCl₃ -CD₃ OD), δ:0.87 (3H, m), 1.05-1.50 (51H, m), 2.82 (4H, s),4.30-4.75 (2H, m), 5.17 (2H, s), 7.33 (5H, s).

Preparation 140 ##STR620##

n-Tetracosanoyl-L-Ala-D-Glu(α-OBzl) (2) was prepared substantially inthe same manner as Preparation 138.

IR (Nujol): 3360, 1740, 1710, 1640 cm⁻¹.

NMR (CDCl₃ -CD₃ OD), δ: 0.88 (3H, m), 1.06-1.70 (47H, m), 2.00-2.50 (4H,m), 516 (2H, s), 736 (5H, s).

Example 165

(1) Step 1 ##STR621##

L-Lys(ε-Z)-D-AlaOH (2) (0.915 g) was dissolved in a mixture of methylenechloride (40 ml) methanol (40 ml) and triethylamine (0.53 g).

To this solution was added n-octanoyl-D-Glu(α-OBzl)-γ-OSu (1) (1.20 g)and the resulting solution was left overnight at ambient temperature.The reaction mixture was evaporated to give a paste, to which were addedwater (50 ml), ether (50 ml) and 1N hydrogen chloride. The resultingmixture was stirred well and ether was evaporated to separate crystalsfrom the aqueous layer. The crystals were filtered, washed with waterand then dried to give n-octanoyl-γ-D-Glu(α-OBzl)-L-Lys(ε-Z)-D-AlaOH (3)(1.50 g).

IR (Nujol): 3300, 1725, 1685, 1650, 1630 cm⁻¹.

NMR (DMSO-d₆): δ0.84 (3H, t, J=7 Hz), 1.00-2.40 (25H, m), 2.84-3.12 (2H,m), 4.10-4.50 (3H, m), 5.00 (2H, s), 5.10 (2H, s), 7.32 (10H, s), 8.10(2H, t, J=8 Hz), 8.84 (1H, d, J=8 Hz).

(2) Step 2 ##STR622##

Octanoyl-γ-D-Glu(α-OBzl)-L-Lys(ε-Z)-D-AlaOH (3) (1.20 g) was dissolvedin acetic acid (50 ml) and hydrogenated over palladium black (150 mg).The catalyst was removed by filtration and the filtrate was evaporatedto give a paste. The paste was allowed to stand to give crystals. Thecrystals were washed throughly with diethyl ether to giveoctanoyl-γ-D-Glu(α-OH)-L-Lys-D-AlaOH (4) (0.80 g). [α]_(D) =+41.7(C=0.2, acetic acid).

IR (Nujol): 3360, 1710 (sh), 1640 cm⁻¹.

NMR (D₂ O): δ0.84 (3H, obscure t, J=7 Hz), 1.00-2.50 (25H, m), 2.80-3.10(2H, m), 4.00-4.40 (3H, m).

EXAMPLE 166

(1) Step 1 ##STR623##

Stearoyl-γ-D-Glu(α-OBzl)-L-Lys(ε-Z)-D-AlaOH (3) was preparedsubstantially in the same manner as step (1) of Example 165.

NMR (DMSO-d₆): δ0.84 (3H, t, J=7 Hz), 1.00-2.40 (45H, m), 2.80-3.10 (2H,m), 4.00-4.80 (3H, m), 5.00 (2H, s), 5.08 (2H, s), 7.32 (10H, s), 7.80(1H, d, J=8 Hz), 8.08 (2H, t, J=8 Hz).

(2) Step 2 ##STR624##

Stearoyl-γ-D-Glu(α-OH)-L-Lys-D-AlaOH (4) was prepared substantially inthe same manner as step (2) of Example 165. [α]_(D) =-11.10 (C=0.21,acetic acid).

IR (Nujol): 3350, 1730, 1640 cm⁻¹.

NMR (NaOD-D₂ O), δ:0.68-2.80 (50H, m), 4.10-4.50 (3H, m).

EXAMPLE 167

(1) Step 1 ##STR625##

Stearoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(1) (2.20 g) was dissolvedin tetrahydrofuran (50 ml), and ethereal diazomethane solution was addedthereto until pale yellow of the solution persisted. After 10 minutes,excess diazomethane was destroyed by adding acetic acid thereto. Theresulting solution was evaporated to give a white crystalline residue,which was washed with diisopropyl ether to givestearoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-(OMe)-(D)-(OMe) (2)(2.10 g).

IR (Nujol): 3300, 1740, 1680, 1640 cm⁻¹.

NMR (CDCl₃), δ:0.83-2.50 (45H, m), 3.71 (3H, s), 3.75 (3H, s), 4.10-5.00(3H, m), 5.20 (2H, s), 7.36 (5H, s).

(2) Step 2 ##STR626##

Stearoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-(OMe)-(D)-(OMe) (2)(0.90 g) was dissolved in a mixture of acetic acid (10 ml) and hydrogenchloride-saturated acetic acid (4 ml). The solution as allowed to standfor 15 minutes at ambient temperature and then evaporated to give acrystalline residue.

The crystalline residue was throughly washed with isopropyl ether togive stearoyl-γ-D-Glu(α-OBzl)-(L)-mesoDAP-(L)-(OMe)-(D)-(OMe)hydrochloric acid salt (3).

IR (Nujol): 3300, 1740, 1640 cm⁻¹.

NMR (DMSO-d₆), δ:0.76-2.40 (45H, m), 3.64 (3H, s), 3.76 (3H, s),3.84-4.40 (3H, m), 5.12 (2H, s), 7.38 (5H, s).

(3) Step 3 ##STR627##

Stearoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-(OMe)-(D)-(OMe) (4) was preparedsubstantially in the same manner as step (2) of Example 165.

[α]_(D) =+3.901 (C=0.2, acetic acid).

IR (Nujol): 3300, 1730, 1640, 1600 cm⁻¹.

NMR (DMSO-d₆), δ:0.70-2.32 (45H, m), 3.62 (3H, s), 3.74 (3H, s),3.80-4.32 (3H, m), 8.02 (1H, d, J=8 Hz), 8.30 (1H, d, J=8 Hz).

EXAMPLE 168

(1) Step 1 ##STR628##

To a suspension of n-docosanoyl-L-Ala-D-Glu(α-OBzl)-γ-OSu (1) (2.91 g)in methylene chloride (50 ml) were added triethylamine (0.89 g) andBoc-(D)-mesoDAP-(L)-D-AlaOH (2) (1.44 g). The mixture was kept for 66hours at ambient temperature and then concentrated in vacuo. To theresidue were added water (100 ml), 1N hydrochloric acid (10 ml) andethylether (30 ml). The resulting mixture was stirred for 15 minutes andethylether was removed. The precipitate was collected by filtration andthen washed with water to give a crude product which was washed withdiisopropylether to given-docosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (3)(3.60 g).

IR (Nujol): 3300, 1720, 1680, 1630 cm⁻¹.

NMR (CDCl₃ -CD₃ OD), δ:0.94 (3H, m), 1.1-2.5 (65H, m), 5.21 (2H, s),7.38 (5H, s).

(2) Step 2 ##STR629##

To a solution ofn-docosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-AlaOH (3)(1.5 g) in a mixture of tetrahydrofuran (40 ml) and chloroform (20 ml)was added 0.6M diazomethane in ethylether (10 ml). The resulting mixturewas stirred for 30 minutes at ambient temperature and then the excessreagent was decomposed with acetic acid. The reaction mixture wasconcentrated and the residue was washed with ethylether and collected byfiltration to given-docosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala(OMe)-(D)-OMe(4) (1.36 g).

NMR (CDCl₃ -CD₃ OD), δ:0.90 (3H, m), 1.1-2.6 (65H, m), 3.71 (3H, s),3.74 (3H, s), 5.19 (2H, s), 7.48 (5H, s).

(3) Step 3 ##STR630##

n-Docosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe-(D)-OMe(4) (1.25 g) was dissolved in acetic acid (20 ml) and hydrogenated underan atmospheric pressure of hydrogen over 10% palladium charcoal. Afterremoval of the catalyst, acetic acid (8 ml) saturated with hydrogenchloride was added to the filtrate. The resulting mixture was kept for 3hours at ambient temperature and concentrated in vacuo to give acrystalline residue, which was collected and washed withdiisopropylether to given-docosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-D-AlaOMe-(D)-OMe hydrochloric acidsalt (5) (1.08 g).

[α]_(D) =-11.42° (C=0.30, acetic acid).

IR (Nujol): 3380, 1745, 1630 cm⁻¹.

NMR (DMSO-d₆), δ:0.87 (3H, m), 1.05-2.45 (56H, m), 3.63 (3H, s), 3.75(3H, s).

EXAMPLE 169 ##STR631##

n-Docosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (2) wasprepared substantially in the same manner as step 3 of Example 168.

[α]_(D) =-19.34° (C=0.20, acetic acid).

IR (Nujol): 3380, 1740, 1630 cm⁻¹.

NMR (NaOD-D₂ O), δ:0.91 (3H, m), 3.25 (1H, m), 4.0-4.6 (5H, m).

EXAMPLE 170

(1) Step 1 ##STR632##

n-Tetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(3) was prepared substantially in the same manner as step 1 of Example168.

IR (Nujol): 3300, 1720, 1680, 1630 cm⁻¹.

NMR (CDCl₃ -CD₃ OD), δ:0.90 (3H, m), 1.05-2.50 (69H, m), 5.15 (2H, s),7.34 (5H, s).

(2) Step 2 ##STR633##

n-Tetracosanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe-(D)-OMe(4) was prepared substantially in the same manner as step 2 of Example168.

IR (Nujol): 3300, 1735, 1685, 1630 cm⁻¹.

NMR (CDCl₃ -CD₃ OD), δ:0.89 (3H, m), 1.05-2.50 (69H, m), 3.69 (3H, s),3.73 (3H, s), 5.18 (2H, s), 7.42 (15H, s).

(3) Step 3 ##STR634##

n-Tetracosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOMe-(D)-OMehydrochloric acid salt (5) was prepared substantially in the same manneras step 3 of Example 168.

[α]_(D) =-14.01 (C=0.26, acetic acid).

IR (Nujol): 3380, 1745, 1630 cm⁻¹.

NMR (DMSO-d₆), δ:0.85 (3H, m), 1.0-2.4 (60H, m), 3.60 (3H, s), 3.72 (3H,s).

(4) Step 4 ##STR635##

D-Tetracosanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (6) wasprepared substantially in the same manner as step 3 of Example 168.

IR (Nujol): 3280, 1730, 1630 cm⁻¹.

NMR (D₂ O+NaHCO₃), δ:0.88 (3H, m), 3.72 (1H, m), 3.95-4.6 (4H, m).

EXAMPLE 171 ##STR636##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe(3) was prepared substantially in the same manner as step 1 of Example168.

IR (Nujol): 3250, 1720, 1680, 1630 cm⁻¹.

NMR (CD₃ OD), δ:1.2-1.9 (17H, m), 2.12 (3H, s), 1.9-2.6 (2H, m), 3.70(6H, s), 3.97 (2H, s), 4.0-4.6 (4H, m), 4.98 (1H, q, J=7 Hz), 5.18 (2H,), 7.35 (5H, s).

EXAMPLE 172

(1) Step 1 ##STR637##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe(3) was prepared substantially in the same manner as step 1 of Example168.

IR (Nujol): 3260, 1735, 1635 cm⁻¹.

NMR (CDCl₃), δ:1.1-2.5 (10H, m), 1.35 (3H, d, J=7 Hz), 1.42 (3H, d, J=7Hz), 2.08 (3H, s), 3.60 (3H, s), 3.67 (3H, s), 3.94 (2H, d, J=7 Hz),4.1-4.8 (4H, m), 4.9-5.2 (1H, m), 5.06 (2H, s), 5.09 (2H, s), 7.30 (10H,s).

(2) Step 2 ##STR638##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOMe-(D)-OMe(3) (1.32 g) was hydrogenated to acetic acid (15 ml) over 10% palladiumcharcoal (0.4 g) for 1.5 hours under an atmospheric pressure of hydrogenat ambient temperature. After completion of the reaction, the catalystwas filtered off and the filtrate was evaporated to dryness. The residuewas pulverized with ether to giveD-Lac(OAc)-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GluOMe-(D)-OMe (4) (1.1g).

NMR (D₂ O), δ: 1.2-2.3 (10H, m), 1.43 (3H, d, J=7 Hz), 1.47 (3H, d, J=7Hz), 3.77 (3H, s), 3.87 (3H, s), 4.03 (2H, s), 4.1-4.5 (4H, m), 5.07(1H, q, J=7 Hz).

EXAMPLE 173

(1) Step 1 ##STR639##

D-LaC(OAc)-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH-(D)-OMe(3) was prepared substantially in the same manner as step 1 of Example168.

IR (Nujol): 3270, 1735, 1650 cm⁻¹.

NMR (CDCl₃), δ:1.2-2.7 (16H, m), 1.43 (9H, s), 2.12 (3H, s), 3.70 (3H,s), 3.8-4.5 (7H, m), 4.9-5.3 (1H, m), 5.13 (2H, s), 7.32 (5H, s).

(2) Step 2 ##STR640##

D-Lac(OAc)-L-Ala-γ-D-Glu(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH-(D)-OMe (4)was prepared substantially in the same manner as step 2 of Example 172.

IR (Nujol): 3300, 1725, 1650 cm⁻¹.

NMR (CD₃ OD), δ: 1.3-2.6 (16H, m), 1.43 (9H, s), 2.11 (3H, s), 3.70 (3H,s), 3.93 (2H, s), 4.2-4.6 (4H, m), 5.08 (1H, q, J=7 Hz).

EXAMPLE 174

(1) Step 1 ##STR641##

Heptanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (1) (1.00 g) wasdissolved in the mixture of water (10 ml) and dioxane (20 ml).Triethylamine (730 mg) and2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (640 mg) was addedto the solution. After stirring for 16 hours at ambient temperature, thereaction mixture was concentrated. To the residue was added waters (30ml), 1N hydrochloric acid (8 ml) and ethyl acetate. The organic layerwas separated and washed with brine, dried over magnesium sulfate andconcentrated in vacuo to give an oily residue, which was treated withethylether and diisopropylether to giveheptanoyl-γ-D-Glu(α-OH)-(L)-Boc(D)-mesoDAP-(L)-D-AlaOH (2)(925 mg).

IR (Nujol): 3300, 1720, 1630 cm⁻¹.

NMR (CD₃ OD), δ: 0.90 (3H, m), 1.1-2.6 (23H, m). 3.70 (1H, t, J=6 Hz),3.90-4.65 (3H, m).

(2) Step 2 ##STR642##

Heptanoyl-γ-D-Glu(α-OMe)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe-(D)-OMe (3)was prepared substantially in the same manner as step 2 of Example 168.

IR (Nujol): 3300, 1735, 1685, 1630 cm⁻¹.

NMR (CD₃ OD), δ: 0.91(3H, m), 1.15-1.90 (16H, m), 2.00-2.45 (4H, m),3.71 (9H, s), 4.06 (1H, m), 4.24-4.56 (3H, m).

(3) Step 3 ##STR643##

Heptanoyl-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-D-AlaOMe-(D)-OMe (4) wasprepared substantially in the same manner as step 3 of Example 168.

IR (Nujol): 3320, 3080, 1740, 1635 cm⁻¹.

NMR (CD₃ OD), δ: 0.92 (3H, m), 1.16-2.46 (26H, m), 3.74 (9H, s),4.21-4.60 (4H, m).

EXAMPLE 175

(1) Step 1 ##STR644##

Octanoyl-γ-D-Glu(α-OMe)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOMe-D-OMe (2) wasprepared substantially in the same manner as step 2 of Example 168.

IR (Nujol): 3370, 1750, 1690, 1640 cm⁻¹.

NMR (CDCl₃), δ: 0.89 (3H, m), 1.05-2.75 (37H, m), 3.72 (9H, s), 4.20(1H, m), 4.35-4.75 (3H, m), 5.35 (1H, d, J=8 Hz), 6.75 (1H, d, J=8 Hz),6.85 (1H, d, J=8 Hz), 7.42 (1H, d, J=7 Hz).

(2) Step 2 ##STR645##

Octanoyl-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-D-AlaOMe-(D)-OMe hydrochloricacid salt (3) was prepared substantially in the same manner as step 3 ofExample 168.

IR (Nujol): 3280, 1735, 1630 cm⁻¹.

NMR (CD₃ OD), δ: 0.89 (3H, m), 1.2-2.5 (22H, m), 3.72 (6H, s), 3.86 (3H,s), 4.07 (1H, t, J=6 Hz), 4.19-4.65 (3H, m).

EXAMPLE 176

(1) Step 1 ##STR646##

D-Lac-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-GlyOMe (3) wasprepared substantially in the same manner as step 1 of Example 168.

NMR (CD₃ OD), δ: 1.38 (6H, d, J=7 Hz), 1.47 (9H, s), 1.3-2.5 (10H, m),3.75 (3H, s), 4.00 (2H, s), 4.0-4.6 (5H, m), 5.20 (2H, s), 7.40 (5H, s).

(2) Step 2 ##STR647##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOme (4) was preparedsubstantially in the same manner as step 3 of Example 168.

IR cm⁻¹ Nujol: 3280, 1740, 1640.

NMR (D₂ O), δ: 1.38 (3H, d, J=7 Hz), 1.41 (3H, d, J=7 Hz), 1.3-2.5 (10H,m), 3.75 (3H, s), 3.98 (2H, s), 4.0-4.6 (5H, m).

EXAMPLE 177

(1) Step 1 ##STR648##

D-Lac-L-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH-(D)-OMe (3)was prepared substantially in the same manner as step 1 of Example 168.

NMR (CD₃ OD), δ: 1.42 (9H, s), 1.2-2.5 (16H, m), 3.70 (3H, s), 3.92 (2H,s), 4.0-4.6 (5H, m), 5.17 (2H, s), 7.35 (5H, s).

(2) Step 2 ##STR649##

D-Lac-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH-(D)-OMe (3) was preparedsubstantially in the same manner as step 3 of Example 168.

IR (KBr): 3700-2500, 1730, 1650 cm⁻¹.

NMR (D₂ O), δ: 1.37 (3H, d, J=7 Hz), 1.40 (3H, d, J=7 Hz), 1.3-2.5 (10H,m), 3.80 (2H, s), 3.83 (3H, s), 4.0-4.6 (5H, m).

Preparation 141

(1) Step 1 ##STR650##

To a mixture of L-Ala-D-Glu(γ-OBzl)OMe hydrochloric acid salt (1)(2.9 g)and triethylamine (0.91 g) in acetonitrile (30 ml) was added D-lacticacid N-hydroxysuccinimide ester (2)(2.53 g) under ice-bath cooling. Themixture was stirred for 1.5 hours at ambient temperature. Afterevaporation of acetonitrile, the residue was dissolved in ethyl acetate(70 ml), washed succesively with 2.5% hydrochloric acid (30 ml×2), water(30 ml), 2.5% sodium bicarbonate (30 ml) and water (30 ml×2), dried overmagnesium sulfate and then evaporated. The residue was chromatographedon silica gel column (60 g) eluting with a mixture of chloroform andethyl acetate (1:1) to give D-Lac-L-Ala-D-Glu(γ-OBzl)OMe (3)(2.23 g).

NMR (CDCl₃), δ: 1.30 (3H, d, J=7 Hz), 1.43 (3H, d, J=7 Hz), 2.0-2.7 (4H,m), 3.30 (1H, broad s), 4.73 (3H, s), 4.0-4.8 (3H, m), 5.13 (2H, s),7.35 (5H, s).

(2) Step 2 ##STR651##

A solution of D-Lac-L-Ala-D-Glu(γ-OBzl)OMe (3)(2.09 g) in methanol (100ml) was hydrogenated over 10 % palladium charcoal (0.6 g). After removalof the catalyst, the filtrate was evaporated under reduced pressure togive D-Lac-L-Ala-D-GluOMe (4)(1.6 g).

NMR (CDCl₃), δ: 1.40 (3H, d, J=7 Hz), 1.43 (3H, d, J=7 Hz), 1.9-2.6 (4H,m), 4.75 (3H, s), 4.18 (1H, q, J=7 Hz), 4.3-4.6 (2H, m).

Preparation 142 ##STR652##

A solution of Z-(L)-Boc-(D)-mesoDAP-(L)-GlyOBzl (1) (3.8 g) in a mixtureof methanol (100 ml) and water (10 ml) was hydrogenated over 10%palladium charcoal (0.18 g). After removal of the catalysts, thefiltrate was evaporated to dryness under reduced pressure. The residuewas pulverized with ether to give Boc-(D)-mesoDAP-(L)-GlyOH (2)(2.10 g).

NMR (CD₃ OD), δ: 1.43 (9H, s), 1.3-2.1 (6H, m), 3.8-4.2 (4H, m).

Preparation 143

(1) Step 1 ##STR653##

A mixture of phosphorus oxychloride (120 mg) and N,N-dimethylformamide(1 ml) was stirred at 25°-30° C. for 3 hours and then was cooled to 0°C. To the solution was added methylene chloride solution (2 ml) of2,3;4,6-di-isopropylidene-2-keto-L-gulonic acid mono hydrate (1)(150mg), keeping the temperature at 0° to 5° C. for 30 minutes.

The resulting solution was cooled at -20° to -30° C. On the other hand,D-Glu(OBzl)(2)(120 mg) was dissolved in a mixture of methylene chloride(100 ml) and bis-trimethylsilyl-acetamide (800 mg). The solution thusprepared was added to the above solution at -20° to -30° C. Thetemperature of the mixture was raised to ambient temperature and thenthe mixture was stirred for 15 hours. The reaction mixture wasevaporated in vacuo and extracted with ethyl acetate. The organic layerwas washed with water (×2) and brine and then dried over magnesiumsulfate. The solvent was evaporated to give an oily residue which wassubjected to silica gel column. The column was eluted with chloroformand methanol (20:1). The solvent of the fraction containing, the objectcompound was evaporated to give2,3;4,6-di-isopropylidene-2-keto-L-gulonoyl-γ-D-Glu(α-OBzl)OH (3)(220mg).

NMR (CDCl₃), δ: 1.33 (3H, s), 1.43 (3H, s), 1.53 (6H, s), 2.10-2.40 (4H,m), 4.15 (2H, s), 4.33-4.66 (3H, m), 4.73 (1H, t, J=7 Hz), 5.16 (2H, s),7.33 (5H, s).

(2) Step 2 ##STR654##

2,3;4,6-Di-isopropylidene-2-keto-L-gulonoyl-D-Glu(α-OBzl)OH (3)(950 mg)and N-hydroxysuccinimide (230 mg) was dissolved in tetrahydrofuran (10ml). To the solution was added dicyclohexylcarbodiimide (410 mg) at 0°C. The reaction mixture was stirred at 0° C. and stood overnight in arefrigerator. The precipitates were filtered and washed withtetrahydrofuran. The filtrate was evaporated to give a viscous oil whichwas pulverized with diethylether to give2,3;4,6-di-isopropylidene-2-keto-L-gulonoyl-D-Glu(α-OBzl)OSu (4)(1.1 g).

NMR (CDCl₃), δ: 1.30 (3H, s), 1.40 (3H, s), 1.50 (6H, s), 2.77 (4H, s),4.10-4.90 (6H, s), 5.16 (2H, s), 7.30 (5H, s).

Preparation 144 ##STR655##

1-N-m-hexyl-1,2,3,4,5,-O-tetraacetyl-D-glucaramide N-hydroxylsuccinimylester (2) was prepared substantially in the same manner as step 2 ofPreparation 143.

NMR (CDCl₃), δ: 0.7-1.1 (3H, m), 1.1-2.0 (8H, m), 2.10 (3H, s), 2.22(9H, s), 2.88 (4H, s), 3.0-3.5 (2H, m), 5.2-6.0 (4H, m), 6.23 (1H, m).

Preparation 145 ##STR656##

Boc-(L)-Z-(D)-mesoDAP-(D)-OEt (2) was prepared substantially in the samemanner as Preparation 129.

NMR (CDCl₃), δ: 1.17 (3H, t, J=7 Hz), 1.40 (9H, s), 1.30-2.0 (6H, m),4.13 (2H, q, J=7 Hz), 4.25 (1H, m), 5.07 (2H, s), 5.30 (1H, broad), 7.25(5H, s), 8.12 (1H, broad s).

Preparation 146 ##STR657##

Boc-(L)-Z-(D)-mesoDAP-(L)-D-AlaOMe-(D)-OMe (2) was preparedsubstantially in the same manner as Preparation 126.

IR (Nujol): 3300, 1740, 1685, 1655 cm⁻¹.

Preparation 147 ##STR658##

To a solution of diZ-mesoDAP-(D)-OMe (1)(37.0 g) in methylene chloride(200 ml) was added thionyl chloride (74 ml) and the mixture was refluxedfor an hour. After evaporation of the solvent, the residue was dissolvedin acetonitrile (200 ml). This solution was added to a mixture ofD-alanine (21.0 g) and sodium carbonate (8.3 g) in a mixture of 0.5Nsodium hydride (400 ml) and acetonitrile (200 ml) under icebath cooling.After stirring for 30 minutes at the same temperature, the mixture wasdiluted with ethyl acetate (150 ml). The aqueous layer was evaporatedand the organic layer was extracted with water (150 ml). The aqueouslayer and the extract were combined, washed with ethyl acetate and thenacidified to pH 4 with 20% hydrochloric acid. This solution was put on acolumn of HP 20 (700 ml). After washing with water, the column waseluted with a mixture of methanol and water (4:1) to giveZ-(D)-mesoDAP-(L)-D-AlaOH-(D)-OMe (2)(20.0 g).

NMR (D₂ O-NaHCO₃),δ: 1.2-2.1 (6H, m), 1.35 (3H, d, J=7 Hz), 3.73 (3H,s), 3.9-4.4 (3H, m), 5.10 (2H, s), 7.07 (5H, s).

Preparation 148 ##STR659##

Z-(D)-mesoDAP-(L)-L-AlaOH-(D)-OEt (2) was prepared substantially in thesame manner as Preparation 147.

NMR (D₂ O-NaHCO₃),δ: 1.22 (3H, t, J=7 Hz), 1.37 (3H, d, J=7 Hz), 1.5-2.1(6H, m), 3.8-4.4 (5H, m), 5.12 (2H, s), 7.40 (5H, s).

Preparation 149 ##STR660##

Z-(D)-mesoDAP-(L)-D-AlaOH-(D)-OEt (2) was prepared substantially in thesame manner as Preparatin 147.

NMR (D₂ O-NaHCO₃),δ: 1.23 (3H, t, J=8 Hz), 1.35 (3H, d, J=7 Hz), 1.1-2.0(6H, m), 3.9-4.5 (5H, m), 5.15 (2H, s), 7.34 (5H, s).

Preparation 150 ##STR661##

Boc-(L)-mesoDAP-(L)-D-AlaOH-(D)-OMe (2) was prepared substantially inthe same manner as Preparation 142.

Preparation 151 ##STR662##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOBzl-(D)-OMe (2) was preparedsubstantially in the same manner as Preparations (102) and (106).

NMR (CDCl₃),δ: 1.42 (9H, s), 1.0-2.1 (9H, s), 3.66 (3H, s), 4.0-4.7 (3H,m), 5.07 (2H, s), 5.10 (2H, s), 7.29 (10H, s).

EXAMPLE 178

(1) Step 1 ##STR663##

To an ice-cooling solution ofL-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOH-(D)-NHNHZ (1)(1.23 g)and triethylamine (0.30 g) in a mixture of acetone (12 ml) and water (2ml) was added a solution of1-N-n-hexyl-2,3,4,5-O-tetraacetyl-D-glucaramide N-hydroxylsuccinimylester (2)(0.84 g) in acetone (12 ml). The mixture was stirred overnight,allowing the temperature of the mixture to reach to ambient temperature.

After evaporation, the residue was added to a mixture of water (50 ml)and ethyl acetate (100 ml).

The organic layer was washed with aqueous sodium bicarbonate (30 ml),water (30 ml), 10% hydrochloric acid (30 ml) and water (30 ml×2) andthen dried over magnesium sulfate. The solvent was removed in vacuo togive1-N-n-hexyl-2,3,4,5-O-tetraacetyl-D-glucaramidyl-L-Ala-γ-D-Glu(.alpha.-OBzl)-(L)-Z-(D)-mesoDAP-(L)-GlyOH-(D)-NHNHZ(3)(1.15 g).

NMR (DMSO-d₆),δ: 0.7-1.0 (3H, m), 1.1-1.7 (19H, m), 1.7-2.3 (2H, m),1.96 (3H, s), 2.00 (3H, s), 2.07 (3H, s), 2.12 (3H, s), 2.8-3.1 (2H, m),3.8-4.5 (10H, m), 5.00 (2H, s), 5.06 (2H, s), 5.10 (2H, s), 5.30 (1H,m), 5.60 (1H, m), 7.34 (15H, s), 7.7-8.2 (5H, m), 8.40 (1H, m), 9.98(1H, s).

(2) Step 2 ##STR664##

1-N-n-hexyl-2,3,4,5-O-tetraacetyl-D-Glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH-(D)-NHNH₂(4) was prepared substantially in the same manner as step 2 of Example165.

NMR (CD₃ OD),δ: 0.8-1.1 (3H, m), 1.1-2.5 (21H, m), 2.03 (6H, s), 2.17(3H, s), 2.23 (3H, s), 3.0-3.6 (2H, m), 3.7-4.8 (6H, m), 5.1-6.0 (4H,m).

(3) Step 3 ##STR665##

To an ice-cooling solution of1-N-n-hexyl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH-(D)-NHNH₂(4)(0.64 g) in a mixture of 10% hydrochloric acid (3 ml) and 1,4-dioxane(6 ml) was added N-bromosuccinimide (0.2 g). After stirring for an hourat ambient temperature, the mixture was treated with sodium bisulfiteand then adjusted to pH 4 with aqueous sodium carbonate. Afterevaporation, the residue was dissolved in 1N hydrochloric acid (2 ml)and put on a column of HP-20 (40 ml). After removal of inorganic saltswith water, the product was obtained by eluting with 70% methanol. Theeluate was lyophilized to give1-N-n-hexyl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH(5) (0.43 g).

NMR (D₂ O), δ: 0.7-1.0 (3H, m), 1.0-1.6 (19H, m), 1.6-2.5 (2H, m), 2.14(3H, s), 2.16 (3H, s), 2.22 (3H, s), 2.24 (3H, s), 3.0-3.3 (2H, m), 3.76(1H, m), 4.0-4.6 (5H, m), 5.1-5.8 (4H, m).

(4) Step 4 ##STR666##

To an ice-cooling solution of1-N-n-hexyl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH(5) (0.33 g) in 50% methanol was added 2N potassium carbonate (2 ml).The mixture was stirred for two hours at the same temperature and thenacidified to pH 4 with 10% hydrochloric acid. After evaporation, theresidue was dissolved in 1N hydrochloric acid (1 ml) and put on a columnof HP-20 (15 ml). Inorganic salts were removed with water and theneluted with 80% methanol. The eluate was lyophilized to give1-N-n-hexyl-D-glucaramidoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH(6) (0.26 g).

IR (Nujol): 3300, 1720, 1640, 1540, 1230 -m⁻¹.

NMR (D₂ O), δ: 0.7-1.0 (3H, m), 1.0-2.5 (21H, m), 3.1-3.5 (2H, m),3.5-4.5 (10H, m).

EXAMPLE 179

(1) Step 1 ##STR667##

D-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-Boc-(D)-mesoDAP-(L)-GlyOH (3) wasprepared substantially in the same manner as step 1 of Example 168.

NMR (CD₃ OD), δ: 1.46 (9H, s), 1.2-2.4 (10H, m), 3.72 (3H, s), 3.93 (2H,s), 4.0-4.6 (5H, m).

(2) Step 2 ##STR668##

D-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-GlyOH (4) was preparedsubstantially in the same manner as step 3 of Example 168.

NMR (D₂ O), δ: 1.40 (3H, d, J=7 Hz), 1.45 (3H, d, J=7 Hz), 1.3-2.6 (10H,m), 3.77 (3H, s), 3.87 (2H, s), 4.2-4.6 (4H, m).

EXAMPLE 180 ##STR669##

D-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-mesoDAP-(L)-GlyOMe (3) was preparedsubstantially in the same manner as steps 1 and 3 of Example 168.

NMR (D₂ O), δ: 1.36 (3H, d, J=7 Hz), 1.42 (3H, d, J=7 Hz), 1.2-2.4 (10H,m), 3.72 (6H, s), 3.98 (2H, s), 4.2-4.5 (4H, m).

EXAMPLE 181

(1) Step 1 ##STR670##

D-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-Boc-(D)-mesoDAP-(D)-OMe-(L)-GlyOH (3) wasprepared substantially in the same manner as step 1 of Example 168.

IR cm⁻¹ Nujol: 3300, 1730, 1660.

NMR (CD₃ OD), δ: 1.47 (9H, s), 1.4-2.5 (16H, m), 3.73 (6H, s), 3.95 (2H,s), 4.0-4.6 (5H).

(2) Step 2 ##STR671##

D-Lac-L-Ala-γ-D-Glu(α-OMe)-(L)-mesoDAP-(D)-OMe-(L)-GlyOH(4) was preparedsubstantially in the same manner as Step 3 of Example 168.

[α]_(D) =22.9°(C=0.2, H₂ O).

IR cm⁻¹ Nujol: 3400, 1745, 1650.

NMR (D₂ O), δ: 1.33 (3H, d, J=7 Hz), 1.40 (3H, d, J=7 Hz), 1.2-2.6 (10H,m), 3.77 (3H, s), 3.83 (3H, s), 4.0-4.6 (5H, m).

EXAMPLE 182

(1) Step 1 ##STR672##

2,3;4,6-diisopropylidene-L-2-ketogulonoyl-γ-D-Glu(α-OBzl)-L-Lys(Z)-D-AlaOH(3) was prepared substantially in the same manner as step 1 of Example165.

IR (Nujol),: 3300, 1750-1620, 1250-1060⁻¹.

(2) Step 2 ##STR673##

2,3;4,6-Diisopropylidene 2-ketogulonoyl-γ-D-Glu(α-OBzl)-L-Lys(Z)-D-AlaOH(3) (0.9 g) was dissolved in dioxane. To the solution was added conc.hydrochloric acid (5 ml), and the mixture was stirred for 20 hours andthen adjusted to pH 6-7 with aqueous sodium bicarbonate solution. Theresultant mixture was concentrated in vacuo and the residue wasdissolved in water. The solution was subjected to carbon column (30 ml).The column was eluted with methanol and ethanol (1:1). The fractionscontaining the object compoud (4) were collected and the solvent wasevaporated to give a foam which was lyophilized to give2-keto-L-gulonoyl-γ-D-Glu(α-OBzl)-L-Lys-D-AlaOH (4) (400 mg).

NMR (D₂ O), δ: 1.32 (3H, d, J=7 Hz), 3.15 (2H, t, J=7 Hz), 3.66-4.50(8H, m), 5.10 (2H, s), 7.43 (5H, s).

(3) Step 3 ##STR674##

2-Keto-L-gulonoyl-γ-D-Glu(α-OH)-L-Lys-D-AlaOH (5) was preparedsubstantially in the same manner as step 2 of Example 165.

NMR (D₂ O), δ: 1.36 (3H, d, J=7 Hz), 3.05 (2H, t, J=7 Hz), 3.70-4.50(8H, m).

EXAMPLE 183

(1) Step 1 ##STR675##

2,3;4,6-di-isopropylidene-2-keto-L-gulonoyl-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH(3) was prepared substantially in the same manner as step 1 of Example165.

IR (Nujol),: 3300, 1730, 1680, 1520, 1250-1060 cm⁻¹.

NMR (CD₃ OD), δ: 1.06 (3H, s), 1.20 (3H, s), 1.47 (15H, s), 4.16-4.50(9H, m), 5.20 (2H, s), 7.40 (5H, s).

(2) Step 2 ##STR676##

2-Keto-L-gulonoyl-γ-D-Glu(α-OBzl)-(L)-mesoDAP-(L)-D-AlaOH (4) wasprepared substantially in the same manner as step 2 of Example 182.

IR (Nujol),: 3300, 1730, 1650, 1530, 1220, 1180, 1050 cm⁻¹.

NMR (D₂ O), δ: 1.33 (3H, d, J=8 Hz), 3.66-4.50 (8H, m), 5.30 (2H, s),7.47 (5H, s).

(3) Step 3 ##STR677##

2-Keto-L-gulonoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (5) was preparedsubstantially in the same manner as step 2 of Example 165.

IR (Nujol),: 3300, 1720, 1670 cm⁻¹.

NMR (D₂ O), δ: 1.38 (3H, d, J=7 Hz), 1.70-2.50 (10H, m), 3.60-4.40 (8H,m).

EXAMPLE 184

(1) Step 1 ##STR678##

Stearoyl-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(D)-OEt-(L)-D-AlaOEt (3) wasprepared substantially in the same manner as step 1 of Example 168.

IR (Nujol): 3280, 1750, 1710, 1650 cm⁻¹.

NMR (CDCl₃),:δ: 0.87 (3H, m), 3.96-4.60 (9H, m), 5.10 (2H, s), 5.13 (2H,s), 7.32 (10H, s).

(2) Step 2 ##STR679##

Stearoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(D)-OEt-(L)-D-AlaOEt(3) (584 mg) was dissolved in acetic acid (20 ml) and hydrogenated under2.5 atmospheric pressures of hydrogen over 10% palladium charcoal. Afterremoval of the catalyst by filtration, the filtrate was evaporated invacuo. To the residue was added hexane to give crystals which werewashed with hot isopropylether to givestearoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-OEt-(L)-D-AlaOEt (4) (340mg).

IR (Nujol): 3290, 1740, 1630 cm⁻¹.

NMR (CF₃ COOH), δ: 0.87 (3H, m), 1.1-2.9 (54H, m). 4.13-5.00 (9H, m).

EXAMPLE 185

(1) Step 1 ##STR680##

Heptanoyl-L-Ala-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(D)-OEt-(L)-D-AlaOEt(3) was prepared substantially in the same manner as Step 1 of Example168.

IR (Nujol): 3260, 1730, 1680, 1620 cm⁻¹.

NMR (CDCl₃), δ: 0.85 (3H, m), 1.05-2.40 (29H, m), 3.93-4.70 (8H, m),5.06 (2H, s), 5.11 (2H, s), 5.58 (1H, d, J=8 Hz), 6.60 (2H, d, J=8 Hz),7.20 (1H, d, J=8 Hz), 7.31 (10H, s).

(2) Step 2 ##STR681##

Heptanoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(D)-OEt-(L)-D-AlaOEt (4) wasprepared substantially in the same manner as step 2 of Example 184.

IR (Nujol): 3280, 1740, 1635 cm⁻¹.

NMR (DMSO-d₆), δ: 0.87 (3H, m), 1.02-2.33 (27H, m), 3.40 (1H, m).

EXAMPLE 186

(1) Step 1 ##STR682##

1-N-Benzyl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OBzl)-(L)-Z-(D)-mesoDAP-(D)-NHNHZ-(L)-GlyOH(3) was prepared in a similar manner to Step 1 of the Example 168.

mp. 176°-178° C.

IR (Nujol): 3350, 1750, 1670, 1530 cm⁻¹.

NMR (DMSO-d₆): δ1.02-2.2 (13H, m), 1.85 (3H, s), 1.94 (3H, s), 2.00 (3H,s), 2.10 (3H, s), 3.6-4.5 (8H, m), 4.95 (2H, s), 5.00 (2H, s), 5.06 (2H,s), 5.1-5.7 (2H, m), 7.20 (5H, s), 7.28 (15H, s), 7.6-8.6 (3H, m). (2)Step 2 ##STR683##

1-N-Benzyl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(D)-NHNH₂-(L)-GlyOH (4) was prepared in a similar manner to the Example 165 Step2.

mp. 178°-181° (dec).

IR (Nujol): 3300, 1750, 1650, 1530 cm⁻¹.

NMR (CH₃ OD): δ1.1-2.5 (13H, m), 1.83 (3H, s), 2.00 (3H, s), 2.13 (3H,s), 2.23 (3H, s), 3.85 (2H, s), 7.25 (5H, s).

(3) Step 3 ##STR684##

1-N-Benzyl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH(5) was prepared in a similar manner to the Example 178 Step 3.

mp. 145°-150° C.

IR (Nujol): 3350, 1760, 1660 (sh), 1650, 1530 cm⁻¹.

NMR (D₂ O): δ1.43 (3H, d, J=7 Hz), 1.3-2.5 (10H, m), 2.00 (3H, s), 2.10(3H, s), 2.23 (6H, s), 3.7-4.0 (3H, m), 4.0-4.5 (3H, m), 5.1-6.0 (4H,m), 7.37 (5H, s).

(4) Step 4 ##STR685##

1-N-Benzyl-D-glucaramidoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH (6)was prepared in a similar manner to the Example 178 Step 4.

mp. 117°-120° C.

IR (Nujol): 3350, 1730, 1640 (sh), 1540 cm⁻¹.

NMR (D₂ O): δ1.42 (3H, d, J=7 Hz), 1.2-2.5 (10H, m), 3.86 (2H, s),3.5-5.0 (10H, m), 7.35 (5H, s).

EXAMPLE 187

(1) Step 1 ##STR686##

1-N-Lauryl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OBzl)-(L)-Z-(D)-mesoDAP-(D)-NHNHZ-(D)-GlyOH(III) was prepared in a similar manner to the Example 168, step 1.

IR (Nujol): 3350, 1760, 1670, 1650, 1540 cm⁻¹.

NMR (DMSOD₆): δ0.9 (3H, m), 1.0-2.3 (33H, m), 1.95 (3H, s), 2.00 (3H,s), 2.07 (3H, s), 2.15 (3H, s), 3.00 (2H, m), 3.2-4.6 (10H, m), 5.08(2H, s), 5.13 (2H, s), 5.18 (2H, s), 5.2-5.8 (2H, m), 7.37 (15H, s),7.7-8.5 (5H, m), 9.20 (1H, m), 9.90 (1H, s).

(2) Step 2 ##STR687##

1-N-Lauryl-2,3,4,5-O-tetraacetyl-(D)-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(D)-NHNH₂-(L)-GlyOH (II) was prepared in a similar manner to the Example 165 step2.

IR (Nujol): 3350, 1760, 1660, 1540 cm⁻¹.

NMR (CD₃ OD): δ0.90 (3H, m), 1.0-2.4 (33H, m), 2.02 (6H, s), 2.13 (3H,s), 2.20 (3H, s), 2.9-3.3 (2H, m), 3.80 (2H, s).

(3) Step 3 ##STR688##

1-N-Lauryl-2,3,4,5-O-tetraacetyl-D-glucaramidoyl-L-Ala-γ-D-Glu(.alpha.-OH)-(L)-mesoDAP-(L)-GlyOH(II) was prepared in a similar manner to the Example 178 step 3.

IR (Nujol): 3350, 1760, 1660, 1540 cm⁻¹.

NMR (D₂ O): δ 0.80 (3H, m), 1.0-2.5 (33H, m), 2.08 (6H, s), 2.16 (3H,s), 2.20 (3H, s), 3.10 (2H, m), 3.78 (2H, s).

(4) Step 4 ##STR689##

1-N-Lauryl-D-glucaramidoyl-L-Ala-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-GlyOH(II) was prepared in a similar manner to the Example 178 step 4.

IR (Nujol): 3300, 1720, 1640, 1540 cm⁻¹.

NMR (D₂ O): δ 0.80 (3H, m), 1.0-2.5 (33H, m), 3.20 (2H, m), 3.80 (2H,m).

Preparation 152 ##STR690##

To a solution of palmitoyl-L-Ala-D-Glu(OH)OBzl (1) (1.84 g) in a mixtureof tetrahydrofuran (20 ml) and chloroform (30 ml) were addedN-hydroxysuccinimide (425 mg) and dicyclohexylcarbodiimide (728 mg). Thereaction mixture was kept for 18 hours at room temperature and theprecipitate was filtered off and washed with chloroform. The filtratewas concentrated in vacuo and the diisopropylether was added to theresidue. The product was collected and dried to affordpalmitoyl-L-Ala-D-Glu(OSu)OBzl (2) (1.70 g).

IR (Nujol): 3300, 1805, 1780, 1745, 1640 cm⁻¹.

NMR (CDCl₃, δ): 0.89 (3H, m), 1.05-2.70 (35H, m), 2.80 (4H, s),4.20-4.83 (2H, m), 5.15 (2H, s), 6.23 (1H, d, J=8 Hz), 7.30 (5H, s).

Preparation 153 ##STR691##

Trifluoroacetic acid (15 ml) was added to Boc-L-Ala-D-Glu-(OH)OBzl (1)(3.26 g) and the mixture was stirred for 20 minutes at room temperature,concentrated in vacuo and washed with diisopropylether. The oil wasdissolved in a mixture of water (15 ml) and sodium bicarbonate was addeduntil the pH of the solution became 8-9. The mixture of water (6 ml) andacetone (20 ml) was added to the solution and palmitoyl chloride (2.20g) was added in one portion. After stirring for 30 minutes at roomtemperature, the pH of the solution was adjusted to 3 with1N-hydrochloric acid and the reaction mixture was concentrated. To theresidue was added water (30 ml) and the precipitates was collected anddried to give a crude product, which was purified by columnchromatography of Silicagel (90 g) with chloroform-methanol (30:1, v/v)as an eluent to give palmitoyl-L-Ala-D-Glu(OH)OBzl (2) (2.00 g). mp.131°-132° C.

[α]_(D) -23.38°(C=0.2, CHCl₃).

IR (Nujol): 3300, 1730, 1700, 1650, 1635 cm⁻¹.

NMR (CDCl₃, δ): 0.88 (3H, m), 1.1-1.75 (31H, m), 2.0-2.5 (4H, m),4.4-4.8 (2H, m), 5.11 (2H, s), 7.34 (5H, s).

Preparation 154 ##STR692##

To a solution of Z-(L)-mesoDAP-(L)-D-AlaOBzl (1) (5.97 g) in a mixtureof water (60 ml), dioxane (60 ml) and triethylamine (2.73 g) was droppeda solution of di-tert-butyl carbonate (3.21 g) in dioxane (18 ml) atroom temperature and the pH of the mixture was adjusted between 9 and 10with triethylamine. After stirring for 1 hour, the mixture was dilutedwith water and ethyl acetate (100 ml) was added. The organic layer wasextracted with water three times and the aqueous layers were combined.The pH of the aqueous layer was adjusted to 4-5 with 5% hydrochloricacid and extracted with ethyl acetate. The extract was washed withbrine, dried over magnesium sulfate and concentrated in vacuo. Theresulting residue was crystallized with diisopropylether to giveZ-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOBzl (2) (5.28 g). mp. -143° C. (dec.).

NMR (CD₃ OD): δ1.33 (2H, d, J=8 Hz), 1.40 (9H, s), 1.2-2.0 (6H, m),3.9-4.2 (2H, m), 4.40 (1H, q, J=8 Hz), 5.05 (2H, s), 5.10 (2H, s), 7.30(10H, s).

Preparation 155 ##STR693##

Z-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOBzl-(D)-NHNHBoc (1) (28 g) was added totrifluoroacetic acid (280 ml) and the mixture was stirred for 1 hour atroom temperature. After evaporation of trifluoroacetic acid, the residuewas dissolved in a mixture of water (240 ml), 1N hydrochloric acid (40ml) and dioxane (200 ml), and N-bromosuccinimide (16.4 g) was added at0° C. After stirring for 0.5 hours at the same temperature, the reactionmixture was treated with an aqueous solution of sodium sulfite andconcentrated in vacuo. To the residue was added 30% aqueous sodiumhydroxide until the pH of the mixture reached to 4-5. The resultingprecipitate was collected and washed with water to giveZ-(L)-mesoDAP-(L)-D-AlaOBzl (2).

IR (Nujol): 3280, 1725, 1685, 1640 cm⁻¹.

NMR (DMSO-d₆): δ 1.34 (3H, d, J=8 Hz), 1.2-2.0 (6H, m), 4.37 (1H, q, J=8Hz), 5.08 (2H, s), 5.16 (2H, s), 7.43 (10H, s).

EXAMPLE 188

(1) Step 1 ##STR694##

Boc-(D)-mesoDAP-(L)-Gly-(D)-NHNHBoc (1) (2.31 g) was dissolved in amixture of dioxane (60 ml) and water (50 ml) and triethylamine (610 mg)and Z-D-Ala-D-Glu(α-OBzl)-γ-OSu (1) (2.70 g) were added. The mixture wasleft for 18 hours at room temperature and concentrated at a reducedpressure. The mixture of water (30 ml), 1N-hydrochloric acid (10 ml) andethyl acetate (100 ml) were added to the residue. The organic layer wasseparated and washed with brine, dried over magnesium sulfate,concentrated in vacuo. The resulting amorphous powder was washed withdiisopropyl ether and collected to give a crude product, which waspurified by silica gel chromatography (100 ml) eluting withchloroform-methanol (10:1-5:1, v/v). The fractions containing the objectcompound (3) were collected and concentrated to dryness to yieldZ-D-Ala-γ-D-Glu-α-OBzl-(L)-Boc-(D)-mesoDAP-(L)-Gly-(D)-NHNHBoc (3) (1.09g). mp -125° C. (dec.). [α]_(D) -4.39 (C=0.21, CHCl₃).

IR (Nujol): 3300, 1720, 1670 cm⁻¹.

(2) Step 2 ##STR695##

Z-D-Ala-γ-D-Glu(α-OBzl)-(L)-Boc-(D)-mesoDAP-(L)-Gly-(D)-NHNHBoc (3) (980ml) was dissolved in acetic acid (20 ml) and hydrogenated under anatmospheric pressure of hydrogen over 10% palladium-charcoal (200 mg).After removal of the catalyst, the filtrate was concentrated at areduced pressure. The resulting syrup was dissolved in water (5 ml) andapplied for HP-20 column chromatography (50 ml) eluting withwater-methanol (1:1, v/v). The fractions containing the object compound(4) were collected and evaporated to dryness to yieldH-D-Ala-γ-D-Glu-(L)-Boc-(D)-mesoDAP-(L)-Gly-(D)-NHNHBoc (4) (610 mg). mp-159° C. (dec.).

[α]_(D) -17.30 (C=0.1, meOH).

IR (Nujol): 3270, 1670 cm⁻¹.

NMR (CD₂ OD): δ1.1-2.4 (31H, m), 3.70-4.55 (6H, m).

EXAMPLE 189

(1) Step 1 ##STR696##

To a solution of Z-(D)-meso-DAP-(L)-D-AlaOH (2) (918 mg) in the mixtureof methylene chloride (25 ml) and methanol (6 ml) was addedtriethylamine (760 mg). After the solution turned clear,palmitoyl-L-Ala-D-Glu(OSu)OBzl (1) (1.61 g) was added to the solution.The mixture was kept for 18 hours at the room temperature andconcentrated in vacuo. To the residue were added water (30 ml) and1N-hydrochloric acid (8 ml). The precipitate was collected, washed withwater and dried to give a crude product (2.06 g), which was washed withhot diisopropylether to givepalmitoyl-L-Ala-γ-D-Glu-(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-D-AlaOH (3) (2.00g). mp 185°-187° C.

[α]_(D) -15.38° (C=0.21, AcOH).

IR (Nujol): 3300, 1725, 1690, 1630 cm⁻¹.

NMR (CDCl₃ -CD₃ OD): δ 0.87 (3H, m), 1.0-2.4 (4H, m), 4.1-4.6 (5H, m),5.06 (2H, s).

(2) Step 2 ##STR697##

Palmitoyl-L-Ala-γ-D-Glu-(α-O-Bzl)-(L)-Z-(D)-mesoDAP-(L)-D-Ala-OH (3)(1.86 g) was dissolved in acetic acid (40 ml) and hydrogenated under anatmospheric pressure of hydrogen over 10% palladium-charcoal (1.0 g).After removal of the catalyst, acetic acid was evaporated off in vacuo.To the residue was added diisopropylether and the crystalline mass wascollected to give palmitoyl-L-Ala-γ-D-Glu-(L)-mesoDAP-(L)-D-AlaOH (4)(1.30 g), mp. 185°-190° C.

[α]_(D) -14.07°(C=0.21, AcOH).

IR (Nujol): 3320, 1730(sh), 1710, 1625 cm⁻¹.

NMR (NaOD-D₂ O, δ): 0.87 (3H, m), 1.05-2.60 (44H, m), 3.91-4.54 (5H, m).

EXAMPLE 190

(1) Step 1 ##STR698##

To the solution ofH-L-Ala-γ-D-Glu-(α-OH)-(L)-Boc-(D)-meso-DAP-(L)-D-Ala-OH (1) (528 mg) ina mixture of methylene chloride (20 ml) and methanol (6 ml) were addedtriethylamine (314 mg) and triacontanoic acid-N-hydroxysuccinimide ester(518 mg). After stirring for 16 hours at room temperature, the reactionmixture was concentrated in vacuo and the mixture of water (10 ml) and1N-hydrochloric acid (4 ml) was added to the residue. The precipitateswere collected, washed with water and dried to give a crude product,which was washed with hot diisopropylether to givetriacontanoyl-L-Ala-γ-D-Glu-(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-AlaOH (2)(610 mg). mp -157° C. (dec.). [α]_(D) -14.67°(C=0.20, AcOH).

IR (Nujol): 3280, 1720, 1625 cm⁻¹.

NMR (CDCl₃ -CD₃ OD, δ): 0.92 (3H, m), 1.1-2.1 (77H, m), 2.15-2.50 (4H,m).

(2) Step 2 ##STR699##

To the suspension oftriacontanoyl-L-Ala-γ-D-Glu-(α-OH)-(L)-Boc-(D)-mesoDAP-(L)-D-Ala-OH (2)(500 mg) in acetic acid (20 ml) was added acetic acid saturated withhydrogen chloride (10 ml) and the mixture was stirred on a steam bathfor 5 minutes and cooled to room temperature and kept for 1.5 hours atthe same temperature. The reaction mixture was concentrated in vacuo andthe residue was washed with water and the precipitates were collectedand dried to givetriacontanoyl-L-Ala-γ-D-Glu-(α-OH)-(L)-mesoDAP-(L)-D-AlaOH (412 mg). mp-185° C. (dec.).

IR (Nujol): 3300, 1720, 1625 cm⁻¹.

NMR (CF₃ CO₂ H, δ): 0.90 (3H, m).

EXAMPLE 191

(1) Step 1 ##STR700##

n-Heptanoyl-γ-D-Glu(α-OBzl)-(L)-Z-(D)-mesoDAP-(L)-D-AlaOEt-(D)OEt (3)was prepared substantially in the same manner as step 1 of Example 189.

mp 141-142° C.

[α]_(D) =-10.31° (C=0.22, CHCl₃).

NMR (DMSO-d₆), δ: 0.85 (3H, m), 1.05-2.40 (29H, m), 3.93-4.70 (8H, m),5.06 (2H, s), 5.11 (2H, s), 5.58 (1H, d, J=8 Hz), 6.60 (2H, d, J=8 Hz),7.20 (1H, d, J=8 Hz), 7.31 (10H, s).

(2) Step 2 ##STR701##

n-Heptanoyl-γ-D-Glu(α-OH)-(L)-mesoDAP-(L)-AlaOEt-(D)-OEt (4) wasprepared substantially in the same manner as step 2 of Example 189. mp.179°-182° C. [α]_(D) =-13.85° (C=0.29, AcOH).

NMR (DMSO-d₆),: 0.87 (3H, m), 1.02-2.33 (27H, m), 3.40 (1H, m).

EXAMPLE 192

(1) Step 1 ##STR702##

Heptanoyl-γ-D-Glu-(α-Obzl)-(L)-Z-(D)-mesoDAP-(D)-OEt-(L)-D-AlaOH (3) wasprepared substantially in the same manner as step 1 of Example 189. mp.94°-102° C. [α]_(D) -1.06 (C=0.21, CHCl₃).

IR (Nujol): 3280, 1735, 1685, 1630 cm⁻¹.

NMR (CDCl₃, δ): 0.86 (3H, m), 1.05-2.55 (26H, m), 3.9-4.9 (6H, m), 5.10(2H, s), 5.16 (2H, s), 7.32 (10H, s).

(2) Step 2 ##STR703##

Heptanoyl-γ-D-Glu-(α-OBzl)-(L)-Z-(D)-mesoDAP-(D)-OEt-(L)-D-AlaOH (3)(930 mg) was dissolved in acetic acid (20 ml) and hydrogenated under anatmospheric pressure of hydrogen over 10% palladium-charcoal (200 mg).After removal of the catalyst, acetic acid was evaporated in vacuo. Theresidue was purified by column chromatography of HP-20 (50 ml) withethanol-H20 (3:7, v/v) as an eluent to giveheptanoyl-γ-D-Glu-(α-OH)-(L)-mesoDAP-(D)-OMe-(L)-D-AlaOH (4) (490 mg).

mp. 218° C.

[α]_(D) -14.63° (C=0.2, H₂ O).

IR (Nujol): 3280, 1740, 1635 cm⁻¹.

NMR (D₂ O, δ): 0.90 (3H, m), 1.2-2.6 (26H, m), 4.1-4.55 (6H, m).

We claim:
 1. A compound of the formula or its salt: ##STR704## whereinR_(a) ² is mono- or di-carboxy (or protected carboxy)-lower alkyl orar(carboxy or protected carboxy)-lower alkyl whose aryl moiety may besubstituted by hydroxy,R_(b) ² is hydrogen or lower alkyl, R^(r) ishydrogen or amino protective group, and R^(q) is carboxy or protectedcarboxy, provided that when R_(b) ² is hydrogen, then R_(a) ² is mono-or di-carboxy lower alkyl having 1 and 3 to 6 carbon atoms, --CH₂ CH₂COOH or ar(carboxy) lower alkyl whose aryl moiety may be substituted byhydroxy.
 2. A compound according to claim 1, whereinR_(a) ² iscarboxymethyl or --CH₂ CH₂ COOH, and R_(b) ² is hydrogen.
 3. A compoundof the formula or its salt: ##STR705## wherein R_(c) ¹ is2-acetoxypropionyl, n is the integer 1, R_(b) ¹ is hydrogen, isopropyl,hydroxymethyl, benzyloxymethyl or benzyl, R^(p) is benzyloxycarbonyl,and m is the integer 2;R_(c) ¹ is 2-acetoxypropionyl, n is the integer1, R_(b) ¹ is methyl, R^(p) is benzyloxycarbonyl and m is the integer 2;R_(c) ¹ is t-butoxycarbonyl, n is the integer 2, R_(b) ¹ is methyl,R^(p) is benzyloxycarbonxyl and m is the integer 2; R_(c) ¹ is2-acetoxypropionyl, hexanoyl, heptanoyl, octanoyl, stearoyl,2-hexadecyloctadecanoyl, 2-docosyltetracosanoyl, phenoxyacetyl,acetylmandelyl, phenylcarbamoyl, O-benzylsalicyloyl, nicotinoyl,O-acetylbenziloyl, diphenylacetyl, cinnamoyl or adamantane-1-carbonyl, nis the integer 0, R^(p) is benzyloxycarbonyl and m is the integer 2; orR_(c) ¹ is heptanoyl or acetylmandelyl, n is the integer 0, R^(p) isbenzyloxycarbonyl and m is the integer 3.